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Application of Thermostable Xylanases at Mashing and During Germination
S. Malfliet, L. De Cooman and G. Aerts

For the evaluation of the intrinsic lautering performance of malt an in-house test, predictive for both lauter tun and mash filter operations, has been used. Commercial thermostable endoxylanases added during mashing differ in their ability to increase the filtration rate. Especially the addition of a family 10 endoxylanase at mashing-in can be very efficient in view of wort filtration. Family 10 endoxylanases degrade barley arabinoxylans in an effective way and are not inhibited by the Hordeum vulgare L. xylanase inhibitor (HVXI) purified from barley. Moreover, the addition of this family 10 endoxylanase during germination has a positive effect on the filtration capacity of the kilned malt because the added endoxylanase activity is only partly destroyed during kilning. Furthermore, addition of endoxylanases during germination appears to have no effect on standard malt quality characteristics.Mash filtration is considered as the rate-limiting step in wort production and, therefore, represents a factor of increasing economic importance. Until today, several parameters that possibly influence mash filtration behaviour are estimated by conventional malt analyses. Factors causing reduced rate of lautering (e.g. viscosity) and thus increased processing time are still poorly characterised [5, 7, 10, 18, 20, 27]. The conventional EBC analysis does not provide sufficient information on malt in terms of mash filtration behaviour [19, 20]. Historically, more research has been done on ?-glucans than on arabinoxylans in regard to wort filtration problems. However, nowadays, it has been observed that the impact of arabinoxylans on viscosity and filterability is at least as important as that of ?-glucans [3, 9, 14, 21, 25]. In particular higher mashing-in temperatures, relevant to brewing circumstances, increase the amount of arabinoxylans released into the wort, resulting in a higher wort viscosity and low rates of wort separation [17]. During malting, enzymes that hydrolyse arabinoxylans are produced late in the germination process, the maximum of endoxylanase activity occurring only after 72 hours of germination [4, 16, 24]. On the other hand, Kuntz and Bamforth showed the development of xylanases during steeping of barley and early in germination [3, 13]. During malting, some arabinoxylans are solubilised from the cell walls but are not extensively degraded by endogenous xylanases [15]. During the mashing process, because of the restricted temperature stability of endogenous xylanases [15], the addition of heat-stable exogenous xylanases was found to be an effective strategy to degrade arabinoxylans, resulting in an increased filtration rate and a reduced mash viscosity [1, 22].An optimal cell wall degradation during malting, in combination with the presence of more thermostable xylanases in the kilned malt can increase the filtration rate. Therefore, since incomplete degradation of the endosperm cell walls also reduces the yield of extract [17], thermostable xylanases were added in this study at mashing-in or during germination in order to investigate their effect on the filtration behaviour of the kilned malt.Two very different malt samples produced on an industrial scale, were used during this study. Malt A (variety Sebastian, 2009, French harvest) is an evenly well modified 2-row spring barley malt, and malt B (variety Cervoise, 2009, French harvest) is an undermodified 6-row winter barley malt with a relatively high percentage of whole unmodified grains. The analytical data of both malt samples are listed in table 1.The malt samples were characterised according to the analytical methods of Analytica EBC [8] for moisture content (4.2), extract (4.5.1), total protein (4.3.1), soluble protein (4.9.1), friability (4.15), partly unmodified grains (4.15), whole unmodified grains (4.15), wort viscosity (4.8), wort colour (4.18) and wort pH (8.17).The in-house filtration test, introduced by a commercial malting plant, was carried out via a modified congress mashing procedure. Fifty grams of malt were milled using a coarse grind (1.5 mm) and two hundred mL of water at 62 ?C were added to the grist. The mash was continuously stirred (100 rpm) and the temperature was maintained at 62 ?C for 115 min (according to the mashing time of an EBC Congress mash). Whereas in the EBC Congress procedure the mash is filtered at 20 ?C, in this test the mash is filtered at 62 ?C. The filtration rate of the mashes was determined by transferring the mashed samples into a fluted filter paper (Whatman 597 ?). After recirculation of the initial wort (about 10 mL), the filtrate was collected. The volume of the filtrate was measured every 5 min during the first quarter and then every quarter of an hour.Endoxylanase activities were determined in two different commercially available enzyme preparations (Enzyme 1 and Enzyme 2) using the colorimetric Xylazyme AX method (Megazyme T-XAX200 10/2008 data booklet), with some adaptations in which xylanase preparations were diluted in maleate buffer (100 mM, pH 5.6) and Xylazyme AX tablets were added after a pre-incubation of 5 min at 40 ?C or 62 ?C. The suspensions were incubated for exactly 10 min at 40 ?C or 62 ?C and the reaction was stopped by the addition of 10.0 mL Trizma base solution (2.0 % w/v, pH 9.0) and vigorous vortex-mixing. After centrifugation, A590 nm of the supernatant was measured against a control, prepared by incubating Xylazyme AX tablets in buffer without enzyme addition. All measurements were performed in triplicate. One xylanase unit is the amount of enzyme required to release one micromole of reducing sugar equivalent (xylose) from arabinoxylan per minute under the conditions of the assay.Endoxylanase activities were also determined after 1 day, 2 days and 5 days of germination and in kilned malt using the same colorimetric Xylazyme AX method. 10.0 g of (finely milled) barley or malt sample were extracted during 30 min in 25.0 mL maleate buffer (100 mM, pH 5.6) at room temperature. After centrifugation, Xylazyme AX tablets were added to the supernatant after a pre-incubation of 5 min at 40 ?C. The suspensions were incubated for 60 min at 40 ?C and the reaction was stopped by the addition of 10.0 mL Trizma base solution (2.0 % w/v, pH 9.0) and vigorous vortex-mixing. After centrifugation, A590 nm of the supernatant was measured against a control, prepared by adding Trizma base solution before the Xylazyme AX tablet. A 6-row winter barley (variety Cervoise, 2009, French harvest) was malted at a 1 kg scale. Steeping was carried out by immersion (wet stage: 7 h at 18 ?C; dry stage: 17 h at 20 ?C; wet stage: 1 h at 18 ?C). Germination was performed in cylindrical drums at a temperature of 20 ?C with continuous moistening of air during the first 2 days and at 18 ?C with moistening of air for 1 min every 3 min during the next 3 days. The drums were slowly rotated for 1 min every 7 min. The germinated barley was kilned using three temperature steps (6 h at 55 ?C, 9 h at 60 ?C and 5 h at 80 ?C).

Descriptors: endoxylanase activity, exogenous xylanases, filtration performance

BrewingScience - Monatsschrift für Brauwissenschaft, 63 (May/June 2010), pp. 133-141