WO2019109160A1

WO2019109160A1 - Composition and method for malt mashing

Info

Publication number: WO2019109160A1
Application number: PCT/BR2018/050450
Authority: WO
Inventors: Mario Tyago MURAKAMI; Mariane NORONHA DOMINGUES; Evandro ANTONIO DE LIMA
Original assignee: Cnpem - Centro Nacional De Pesquisa Em Energia E Materiais
Priority date: 2017-12-08
Filing date: 2018-12-06
Publication date: 2019-06-13
Also published as: BR102017026595A2

Abstract

The present invention relates to an enzyme composition that has hydrolytic activity on polysaccharides present in malt and also to a method for malt mashing for manufacturing drinks, especially beers, involving incubation thereof with said enzyme composition.

Description

COMPOSITION AND METHOD FOR MALT SHOWING

FIELD OF THE INVENTION

The present invention relates to a composition directed to the hydrolysis of polysaccharides of malt and to the method of blending thereof for the manufacture of beverages. More specifically, the present invention comprises a defined enzymatic composition and a malt blot method which involves incubating the malt with said composition. The present invention has application in the food industry, especially in breweries.

BACKGROUND OF THE INVENTION

[002] The current industrial process for brewing is based essentially on the same principles between breweries. The basic ingredients used in its production are treated water, with corrected pH and ionic composition, malt, hops, yeast of the genus Saccharomyces, as well as, possibly, other sources of carbohydrates called "adjuncts", which are employed in different proportions and can be selected from corn, rice or high maltose syrup, for example.

In the process, the barley malt is initially milled. After addition of water and correction of pH and ionic strength, the beginning of the maceration stage, in which the hydrated milled malt is subjected to agitation with gradual increase of temperature, in ramps with defined period of time, allowing the action of the various enzymes of the malt itself (endogenous): b-glucanases (around 45 ° C); proteases (about 52 ° C); b-amylases (about 60 ° C), and - amylases (around 70 ° C). At 77 ° C, these endogenous enzymes are inactivated. Due to the great variety in the quality of the commercial malt as well as its price, enzymatic preparations can be added which may contain α-amylases, β-glucanases, cellulases and hemicellulases to promote the extraction of carbohydrates independently of the enzymatic content present in the grain germinated. After maceration, the product obtained, called wort, is filtered. The addition of enzyme preparations also has the purpose of reducing their viscosity and facilitating filtration. In the use of corn grit as adjuvant, it is liquefied, that is, subjected to a specific enzymatic treatment with commercially available thermostable α-amylases for the degradation of the main component of glucose starch. The liquefied adjunct is then mixed with the must still in the maceration stage.

After filtration, the insoluble material containing unhydrolyzed malt is discarded. The wort is then added to the bitter hop (richer in acids) and boiled for 50 minutes. More hops are added, now with aroma, and the boil continues for another 10 minutes. The purpose of the boil is to sterilize the liquid, extract hops components, eliminate unwanted volatile components and reduce volume of water when necessary. Thereafter the wort rests for 20-30 minutes to promote separation of the trub, mixture of precipitated proteins and other insoluble components that will be removed in a hydrodynamic coolant settler, also known as Whirlpool or Rotapool. The must is then cooled and oxygenated to finally receive the yeast and start the fermentation process. In this In the step, proline-specific endopeptidase-containing and a-acetolactate decarboxylase preparations may be added for the purpose of turbidity and diacetyl control, respectively. The fermentation occurs for 5-7 days at low temperatures (12-14 ° C, with Saccharomyces sp.), Followed by a maturation period at 0 ° C for 3-7 days, variable as a function of the concentration of diacetyl found after the fermentation. Following maturation, the beer is filtered, carbonated, diluted, undergoes the addition of anti-oxidant foam stabilizer and then is emptied.

One point that can be improved in the current process is that of utilizing the malt, reducing the amount of unhydrolyzed malt by means of more efficient exogenous enzymatic compositions. In this sense, WO09805788 describes a process for the production of alcoholic beverages to which is added a mixture of enzymes, the composition of which comprises at least the use of an endoxylanase, an arabinofuranosidase, a -amylase, an endopeptidase, a 1-3 (4) -b-glucanase and may further contain a saccharifying amylase and / or an exopeptidase. W02015032850 comments on the creation of a method for reducing the viscosity of the must by the addition of an arabinofuranosidase from the GH43 family. WO2005118769 discloses an enzymatic composition containing a T. reesei endoglucanase and an Aspergillus-derived GH10 xylanase, which is useful in the grinding and filtration step of the brewing process. EP3017706 discloses methods and enzymatic compositions for the preparation of malted cereals and their use in the manufacture of foods and beverages, mentioning the use of xylanases combined with glucanases for the hydrolysis of malt.

[007] Although there are several proposed compositions for performing malt blast, maintaining activity for the longest possible time of the process is a fundamental feature. Even within a group of enzymes that have the same activity, there are differences in speed, catalytic efficiency and resistance to the conditions of the hydrolysis process. Moreover, not all enzymes having the same catalytic activity will show synergy with the other enzymes that constitute an enzymatic composition: in most of the associations, the effect is null or even negative, reducing the effectiveness of the composition as a whole. Taking into account the question of the temperature ramp conventionally employed for carrying out the malt blast step, it is important that the enzymatic composition may exhibit activity within the stipulated range to ensure maximum process efficiency.

Therefore, compositions containing an enzyme complex capable of closing the malt-fermentation cycle with enhanced efficiency are lacking, maintaining their activity at the process temperature for the longest period and with the minimum enzyme loading possible without intervening in the subsequent fermentation process of the wort since, depending on the set of enzymes employed in the flushing, inhibitory compounds may be released in the medium in order to reduce the overall efficiency of brewing beer or other cereal fermented beverages.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is a composition and a malt-blending method with said composition. More specifically, the present invention is an enzymatic composition comprising the sequence arabinofuranosidase defined as SEQ ID NO: 1, the xylanase sequence defined as SEQ ID NO: 2 and beta-glucanases, in addition to the malt-blotting method with the composition of the present invention.

The present invention has the advantage of being active under the most drastic process conditions, allowing an increase in Brix of the wort and therefore of hydrolysis yield, in addition to promoting a reduction of its viscosity. Furthermore, the present invention has the advantage of being able to positively affect the fermentation of the wort, providing a reduction in the number of dead cells and, consequently, enabling a greater reuse of the yeast employed.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a comparative graph of the performance of the enzymes in synergy seeking the best combination with the lowest possible number of enzymes. Mixed E contains 9 enzymes: SEQ ID NO: 1, Gluc_GH16, b-Gluc1 GH1, O-Xyl_GH31, b-Cg1_OH39, Exo_Xeg_GH5, AraXyl_GH5, Man_GH5 and SEQ ID NO: 2. Mixed contains 6 enzymes: the above minus -Xyl_GH31, Exo_Xeg_GH5 and Man_GH5. Mixed U contains 5 enzymes: the former minus Gluc_GH16. Mixed Y contains 4 enzymes: the above minus SEQ ID NO: 1. Mixed T contains 5 enzymes: SEQ ID NO: 1, Gluc_GHI6, b-Cg1_OH39, AraXyl_GH5 and SEQ ID NO: 2. Mixed Z contains 4 enzymes: the above less Gluc_GH16. Mixed AB contains 3 enzymes: the former less b-Cg-OH39. Mixed AE contains 4 enzymes: SEQ ID NO: 1, Gluc_GH16, b-Xyl_GH39 and SEQ ID NO: 2. Mixed AF contains 3 enzymes: the above less b-Cg1_OH39. Mixed AA contains SEQ ID NO: 1, SEQ ID NO: 1 and SEQ ID NO: 2. Beside each combination was plotted its respective control indicating the contribution, in activity, that each enzyme would present if it were acting alone. The values of the synergic reactions presented are the mean and the standard deviation.

Figure 2 shows a graph with the concentration curves of the enzymes arabinofuranosidase SEQ ID NO: 1 and xylanase SEQ ID NO: 2 acting synergistically on malt bagasse with gritz from a process with addition of the enzyme Laminex® Super 3G and on malt cake with high maltose syrup from a process with the addition of the commercial enzyme Bioglucanase® GB. The experiments were carried out on optimized temperature ramps. Values represent mean and standard deviation.

In Figure 3 the results of the parameters evaluated in the "Mosto Congress" for testing the enzymes SEQ ID NO: 1 and SEQ ID NO: 2 with malt and addition of the commercial enzyme Bioglucanase® GB are presented. In the first graph are plotted the density values in degrees Plato (° P). In the second graph are the yield values, in%. In the third graph are the concentration values of b-glucans remaining in the must, in mg / L. In the fourth graph are plotted the viscosity values in mPa.s. White: malt with addition of Bioglucanase® GB; SEQ ID NO: 2: reaction with addition of 0.6 mg / ml of SEQ ID NO: 2; SEQ ID NO: 1: reaction with addition of 0.6 mg / ml of SEQ ID NO: 1; SEQ ID NO: 1 + 2: the reaction with addition of SEQ ID NO: 1 and SEQ ID NO: 2 or 3 at 0.6 mg / ml each. The commercial enzyme Bioglucanase® GB was added under all conditions at the standard dosage of 150 g / tonne of malt. Values represent mean and standard deviation.

[014] Figure 4 shows the results obtained for the parameters evaluated in the "Mosto Congress" for testing the enzymes SEQ ID NO: 1 and SEQ ID NO: 2 with malt and addition of the commercial enzyme Rohalase® Barley L. In the first graph are plotted the density values in degrees Plato (° P). In the second graph are the yield values, in%. In the third graph are the concentration values of b-glucans remaining in the must, in mg / L. In the fourth graph are plotted the viscosity values in mPa.s. Pure malt: malt only, without addition of any enzyme; SEQ ID NO: 2: reaction with addition of 0.6 mg / ml of SEQ ID NO: 2; SEQ ID NO: 1: reaction with addition of 0.6 mg / ml of SEQ ID NO: 1; SEQ ID NO: 1 + 2: reaction with addition of SEQ ID NO: 1 and SEQ ID NO: 2 at 0.6 mg / ml each. The commercial enzyme Rohalase® Barley L was added under all conditions except pure malt at the standard dosage of 75 g / tonne of malt. Values represent mean and standard deviation.

Figure 5 shows the results of the parameters evaluated in the "Mosto Congress" in the test of enzymes SEQ ID NO: 1 and SEQ ID NO: 2 with malt and addition of commercial enzyme Laminex® MaxFlow 4G. In the first graph are plotted the density values in degrees Plato (° P). In the second graph are the yield values, in%. In the third graph are the concentration values of b-glucans remaining in the must, in mg / L. In the fourth graph are plotted the viscosity values in mPa.s. Pure malt: malt only, without addition of any enzyme; SEQ ID NO: 2: reaction with addition of 0.6 mg / ml of SEQ ID NO: 2; SEQ ID NO: 1: reaction with addition of 0.6 mg / ml of SEQ ID NO: 1; SEQ ID NO: 1 + 2: reaction with addition of SEQ ID NO: 1 and SEQ ID NO: 2 at 0.6 mg / ml each. The commercial enzyme Laminex® MaxFlow 4G was added under all conditions except pure malt at the standard dosage of 75 g / tonne of malt. The values presented represent the mean and the standard deviation.

Figure 6 shows a comparison of arabinofuranosidase SEQ ID NO: 1 (indicated as Abf) and xylanase SEQ ID NO: 2 (indicated as Xyl) added with commercial b-glucanases. All the evaluated parameters were plotted in percentage, and the values of the commercial enzymes were considered as 100%. Bioglucanase, Rohalase, laminex: addition of only each of these commercial enzymes in their standard dosage. B + Xyl + Abf: addition of the commercial enzyme Bioglucanase, of SEQ ID NO: 1 and SEQ ID NO: 2, both SEQs at 0.6 mg / ml; R + Xyl + Abf: addition of the commercial enzyme Rohalase® Barley L and the enzymes SEQ ID NO: 1 and SEQ ID NO: 2; I + Xyl + Abf: addition of the commercial enzyme Laminex® MaxFlow 4G and of the enzymes SEQ ID NO: 1 and SEQ ID NO: 2. The values represent the mean and the standard deviation.

Figure 7 shows test results in the "Mosto Congress" to check the effect of concentration of the commercial enzymes of SEQ ID NO: 1 and SEQ ID NO: 2 on the malt. The commercial enzymes Bioglucanase® GB, Rohalase® Barley L and Laminex® MaxFlow 4G were tested at the concentration of 1.2 mg / ml and compared with the previous test at the standard dosage. Pure malt: malt only, without addition of any enzyme; Bioglucanase + GB in the standard dosage (150 g / tonne malt) plus addition of SEQ ID NO: 1 + SEQ ID NO: 2, both at 0.6 mg / ml. Values represent mean and standard deviation.

Figure 9 shows the results of the "Mosto Congress" evaluation test of the enzymes SEQ ID NO: 1 and SEQ ID NO: 2 as substituents of the commercial b-glucanases. Pure malt: malt only, without addition of any enzyme; Bioglucanase (150 g / ton): reaction with the addition of the commercial enzyme Bioglucanase® GB at the standard dosage of 150 g / tonne of malt; SEQ ID NO: 1 + 2: reaction with addition only of SEQ ID NOs: 1 and 2 (both enzymes at 0.6 mg / mL); Bioglucanase (150 g / ton) + SEQ ID NO: 1 + 2: reaction with addition of commercial enzyme Bioglucanase® GB at the standard dosage of 150 g / tonne malt plus SEQ ID Nos 1 and 2 (at 0.6 mg / ml ). Values are means ± standard deviation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is an enzymatic composition comprising the sequence arabinofuranosidase defined as SEQ ID NO: 1, the xylanase sequence defined as SEQ ID NO: 2 and beta-glucanases.

Arabinofuranosidase is understood to be an enzyme which acts on non-reducing ends of arabino-oligosaccharides and polysaccharide side chains containing arabinose residues joined by bonds of the C 1 -1, -1,3 and / or -1 types , 5.

[021] Xylanase is understood to be the enzyme responsible for the cleavage of b-1,4-type bonds between the xylose residues in the xylan and heteroxylan backbone. Beta-glucanases are understood to be the enzymes responsible for the cleavage of the b-1,3 and b-1,4 type bonds between the glucos residues in glucans. The beta-glucanases of the composition of the present invention may be selected from the group comprising the glycosidic hydroxyl families GH5, GH6, GH7, GH12, GH16, GH17, GH74 or combinations thereof. The beta-glucanases of the composition of the present invention may be derived from, but not restricted to, bacteria or fungi, these beta-glucanases being naturally or heterologous in origin, with or without mutations, whether or not they are mutated.

[023] Compared to three base compositions composed of beta-glucanases, the composition of the present invention allows, under the most drastic process conditions, a mean reduction in the concentration of beta-glucans greater than 50%.

[024] The composition may be presented as a solution or as a solid. In the latter case, the composition may be in the form of freeze-dried powder or granules. The addition of a stabilizer and / or a preservative to the composition is optional.

The present invention further relates to a malt-blasting method characterized in that the malt-comprising solution is incubated with an enzyme composition comprising the sequence arabinofuranosidase defined as SEQ ID NO: 1, the xylanase of defined sequence as SEQ ID NO: 2 and beta-glucanases.

[026] Again, the beta-glucanases of the composition of step (c) may be selected from the group comprising the glycosidic hydrolases families GH5, GH6, GH7, GH12, GH16, GH17, GH74 or combinations thereof. Furthermore, the beta-glucanases of the composition of step (c) may be from bacteria or fungi but are not restricted thereto, these beta-glucanases being of natural or heterologous origin, with or without mutations, whether or not they are derived from a directed mutation .

The incubation referred to in the method of the present invention may include subjecting the malt solution, which solution comprises water, malt and, optionally, pH and ionic strength correction agents, to stirring with gradual increase of temperature, ramps and ramps with defined time periods according to the practices already known in the state of the art.

[028] Example: Malt bag is the name given to the remaining non-hydrolyzed material of the malt bag. It is rich in cellulose (17%) and mainly hemicelluloses (39%), especially arabinoxylans (Valverde, 1994; Bartolomé et al., 2002; Mandalari et al., 2005; Mussato et al., 2006; Robertson et al. , 2010).

From this information 22 recombinant enzymes were selected, among cellulases and hemicellulases, with potential application in the process of malting of the malt to compose a personalized enzymatic cocktail that allowed a greater fractionation of arabinoxylans and glucans and, consequently, an increase in productivity and the reduction of costs of breweries.

The recombinant enzymes that have been tested and the family of the glycosidic hydrolases to which they belong are described in Table 1.

[031] These 22 recombinant enzymes were expressed from (Ni-NTA Agarose-Qiagen) and evaluated in hydrolysis reactions against malt bagasse with high maltose syrup or syrup under the conditions of the flask normally employed by the breweries.

[032] Table 1: recombinant enzymes tested

These enzyme hydrolysis experiments were performed in 1.5 mL microtubes containing 55 mg malt bagasse, 125 μl distilled water, 225 μl 250 mM sodium acetate buffer pH 5.5 and 150 μl enzyme a be tested. The hydrolysis reactions were incubated in a stirrer with temperature control, the agitation being 700 rpm and the temperature increase performed gradually according to the brew ramps used in the breweries. In these tests two temperature ramps were used, one standard call starting at 45 ° C and the other called the optimized one, starting at 60 ° C. Both temperature ramps were closed at 77 ° C. At the end of the process, the samples were centrifuged for 2 minutes at 14,000 x g for sedimentation of the particulate material. The obtained supernatants were used for the quantification of reducing sugar released in the hydrolysis reactions by the colorimetric method with 3,5,5-dinitrosalicylic acid reagent - DNS (Miller, 1959).

Of the 22 enzymes tested under the malt bagasse on the two temperature ramps, at least one enzyme representing each type of activity was selected to be evaluated in synergy experiments. These tests aimed to find the best combination with the smallest possible number of enzymes to make an enzymatic cocktail. In the synergy experiments the hydrolysis reactions were prepared as described above, with the only difference that the enzymes used were diluted 2.5-fold as compared to the activity tests performed for each of them alone. Negative control reactions were also prepared, that is, without the addition of any enzyme. In each enzymatic combination performed, one enzyme activity was excluded at a time, in order to obtain a mixture with a minimum amount of enzymes that presented a considerable gain in bagasse hydrolysis. The quantification of reducing sugar produced in the reactions was determined by the DNS reagent method (Miller, 1959).

[036] The results obtained in the enzyme combination experiments during the synergy tests are shown in Figure 1. The absorbance value obtained in the enzymatic hydrolysis reactions was subtracted from the absorbance value of the respective whites (reactions performed under the same conditions but without addition of enzymes).

It has been found that with 9 enzymes acting in synergy (mixed E-SEQ ID NO: 1 (AbfD3), an endo-glucanase, a b-glucosidase, an α-xylosidase, a b-xylosidase, a xyloglucanase, a arabinoxylanase, a mannanase and SEQ ID NO: 2 (XyllOB) yielded an average value of 11.3 absorbance units (ua). Reducing to 6 enzymes (mixed O-SEQ ID NO: 1, an endo-glucanase, a b-glucosidase, a b-xylosidase, an arabinoxylanase and SEQ ID NO: 2) total activity was for 10.7 5 enzymes (mixed T-SEQ ID NO: 1, an endo-glucanase, a b-xylosidase, an arabinoxylanase and SEQ ID NO: 2) gave an average value of 9.9 water, considering the standard deviation, this reduction in activity from 9 to 5 enzymes was not significantly relevant. With 3 enzymes (mixed AF - SEQ ID NO: 1, one endo-glucanase and SEQ ID NO: 2) the mean activity was 8.5 ua, whereas with only 2 enzymes (mixed AC - SEQ ID NO: 1 and SEQ ID NO: 2) activity was 7.4 ua and again, considering the standard deviation between 5, 3 and 2 enzymes, this variation in mean activity was not significant. Thus, the best cost-benefit ratio, relative to the level of remaining unhydrolyzed material in the bagasse and the amount of enzymes required for such hydrolysis, was with only 2 enzymes, arabinofuranosidase SEQ ID NO: 1 and xylanase SEQ ID NO : 2.

[038] A microscale evaluation of the concentration of the selected enzymes was also carried out, in order to obtain the best concentration of arabinofuranosidase and xylanase in the enzymatic cocktail. In this evaluation enzymatic hydrolysis reactions were prepared using the enzyme arabinofuranosidase SEQ ID NO: 1 at a fixed concentration of 0.6 mg / mL and xylanase concentration variations SEQ ID NO: 2, from 0 to 1.2 mg / mL. These hydrolysates were prepared as described above using bagasse and bagasse with high maltose syrup under the optimized temperature ramp conditions.

[039] Figure 2 shows the results of this evaluation. It was found that at concentrations above 0.6 mg / mL xylanase no significant gains in sugar release were obtained. It was also observed that the behavior of the two enzymes in synergy was very similar against the two types of bagasse tested, from production processes with the addition of different commercial enzymes, indicating that the performance of these enzymes was effective even with variations in the type of bagasse used.

[040] Subsequently, the arabinofuranosidase enzymes, sequence defined as SEQ ID NO: 1, and xylanase, of sequence defined as SEQ ID NO: 2, were expressed in large scale and purified for use in malt "Mosto Congress" equipment (Brassagem Bath - Flow Technology). This equipment allows the analysis of the processing of the wort, making it possible to reproduce from the milling of the malt mixed with heated water until the maceration stage and simulates the temperature ramp.

[041] The hydrolysis reactions performed on the micro reactors of the "Mosto Congress" equipment were prepared using a final volume of 100 mL. These reactions contained 12.5 g of freshly ground mill malt (Bilehler), CaCl2 to a final concentration of 0.3 mg / mL and the commercial enzyme Bioglucanase® GB at standard dosage recommended by the manufacturer (150 g / tonne of malt), in which case 1.8 μl of the enzyme was used. The required amounts of the enzymes SEQ ID NO: 1 and 2 were added to the reaction mixture until a concentration of 1.2 mg / mL (0.6 mg / mL xylanase + 0.6 mg / mL arabinofuranosidase) and water distilled to completion of the reaction volume of 100 mL. Control reactions were also prepared without addition of any enzyme, as well as reactions containing only the enzyme Bioglucanase® GB, reactions containing the enzyme Bioglucanase® GB with the addition of xylanase SEQ ID NO: 2 at concentration of 0.6 mg / mL and Bioglucanase® GB enzyme with the arabinofuranosidase SEQ ID NO: 1 also at the concentration of 0.6 mg / ml. These hydrolysis reactions were performed under agitation at 100 rpm and using optimized temperature ramp conditions.

After the temperature ramp was completed, the reactions were maintained at room temperature, the volumes were set according to their initial values (to correct the volume of water evaporated during the temperature rise) and the result was evaluated immediately. The parameters evaluated were filtration time, b-glucan concentration, densitometry and viscosity. The filtration time consists of manually counting the time required for the particulate material to separate from the liquid through a paper filter with visual observation by the operator. The concentration of b-glucans is measured in a spectrophotometer, at the wavelength of 550 nm, in 3 ml plastic cuvettes. To this end, the filtrate from the flask is incubated for 30 minutes with solution A of the Enzitec ™ Color Gluca Test® kit. As a negative control, this solution is incubated with equal volume of water. The obtained values of b-glucan concentration are in mg / L, having as reference a standard curve. Filtration time and b-glucan concentration data can be used to calculate the efficiency of a given enzyme added in the process.

[043] Brix grades or Plato grades were measured in densimeter (Anton Paar DMA 4,500 Density Meter). These measurements were performed at 20 ° C with approximately 50 mL of sample (filtered liquid from the flask). The first two measurements were made with water and the readings were sequential and automatic. [044] The last parameter evaluated was the viscosity of the samples, measured in a viscometer (Brooksfield Viscometer DV-I Prime), the readings being carried out at 20øC for 10 minutes, yielding an average value in mPa.s.

[045] This same malt hydrolysis test on the "Mosto Congress" equipment was performed with other commercial enzyme cocktails which also have reported b-glucanase activity. Instead of Bioglucanase® GB the enzymes Rohalase® Barley L (AB Enzymes) and Laminex® MaxFlow 4G (Danisco) were used. These two enzymes were used in the standard dosage recommended by the manufacturer of 75 g / tonne of malt.

As can be seen in Figures 3, 4, 5 and 6, the use of the enzymes SEQ ID NO: 1 and 2 in combination with different commercial b-glucanases promoted the increase in the Brix value and in the yield of malt hydrolysis , as well as reduced the concentration of remaining b-glucans in the must and also decreased its viscosity. The average gain with the composition of the present invention for Brix was 3.9% and, for yield, 4.43%. The mean reduction in b-glucan concentration was 55.92% and, at the viscosity, 2.72%.

[047] Experiments were also conducted to evaluate whether the increase in the dosage of the commercial enzymes Bioglucanase® GB, Rohalase® Barley L and Laminex® MaxFlow 4G would result in a result similar to that obtained with the addition of the two enzymes in the enzymatic hydrolysis of the malt. In these tests, hydrolysis reactions were prepared on the "Mosto Congress" equipment using the commercial enzymes in the same final concentration as the others, that is, 1.2 mg / mL in the reactions instead of the concentrations recommended by the Providers. To that end, the concentration of the commercial enzymes was determined by reading the absorbance at 280 nm on the NanoDrop 2000c equipment of Thermo Scientific ™. The same pattern of previous tests with respect to temperature ramp, reaction volume and agitation pattern were adopted in this evaluation.

[048] As can be seen in Figure 7, the increase in concentration of the three commercial enzymes was not effective. This result indicates that the two enzymes SEQ ID NO: 1 and SEQ ID NO: 2 have a complementary role in the hydrolysis of malt, acting on targets different from those of the commercial enzymes, such that a more efficient hydrolysis is independent of the increase in the concentration of the b-glucanases tested.

Another analysis was made with respect to the role played by the addition of the enzymes of the composition of the present invention to the malt in the blunting process, i.e. whether the two enzymes of said composition would be acting with complementary or substituent functions to the commercial enzymes. For this test, hydrolysis was performed using SEQ ID NO: 1 and SEQ ID NO: 2 at 0.6 mg / mL in the presence or not of Bioglucanase® GB at the standard dosage of 150 g / tonne of malt, following the same standard of previous tests carried out on the "Mosto Congress" equipment.

The results obtained in this test, shown in Figure 8, demonstrate that SEQ ID NO: 1 and SEQ ID NO: 2 have an activity enhancing effect of the commercial enzyme preparation containing b-glucanases more pronounced than as a substituent thereof in the process of drilling. [051] Microfermentation tests of the must obtained from the malt with the enzymes arabinofuranosidase SEQ ID NO: 1, xylanase SEQ ID NO: 2 and beta-glucanases were performed. For this test, the flask was made as previously described in the "Mosto Congress" equipment, but using a volume of 400 mL in each flask. The control reactions contained only malt, without addition of any enzyme; in the white BG reactions only the commercial enzyme Bioglucanase® GB was added at the standard dosage of 150 g / tonne of malt, whereas in the reactions of interest there was addition, in addition to the commercial enzyme Bioglucanase® GB, of 0.6 mg / ml of each enzyme purified, maintaining the stoichiometry of 1: 1 between SEQ ID NO: 1 and SEQ ID NO: 2. The blast was performed on the optimized temperature ramp. After the temperature ramp was completed, the reactions were kept at room temperature, the volumes were set according to their initial values and the first parameter evaluated was the filtration time, followed by determination of the b-glucan concentration, densitometry and viscosity.

[052] After evaluation of the blast step, a portion of the filtered must (200 mL) was incubated with 15 grams of yeast under agitation of 148 rpm at room temperature for 24 hours. The resulting material was filtered through a paper filter to separate the yeast and analyzed in the Alcolyzer Beer (Anton Paar) system. This system of analysis provides the values of original extract (in ° P) and apparent extract (in%), density in g / cm3, alcohol content in%, number of dead cells, vitality and a value of the degree of fermentation Apparent Degree of Fermentation). [053] Table 2 presents the results obtained in the microfermentation test. The values represent the means and the standard deviation. Malt refers to must coming from a malt-containing mash, without addition of any enzyme; White BG refers to the musts coming from the fermentation with addition of the commercial enzyme preparation Bioglucanase® GB at the standard dosage (150 g / tonne malt); BG + SEQ ID NO: 1 and SEQ ID NO: 2 (1: 1) = addition of

Bioglucanase® GB at the standard dosage and the enzymes SEQ ID NO: 2 and SEQ ID NO: 1, both at 0.6 mg / ml.

[054] Table 2: microfermentation results

[055] The addition of the enzymes SEQ ID NO: 1 and SEQ ID NO: 2 led to a considerable increase in apparent and original extract values and, in addition, resulted in a 55.25% reduction in the number of dead cells in with no addition of enzymes (malt only), thus allowing more cycles of re-use of the added yeast.

Claims

A malt-blending composition comprising the arabinofuranosidase of the sequence defined as SEQ ID NO: 1, the xylanase of sequence defined as SEQ ID NO: 2 and beta-glucanases.

Composition according to claim 1, characterized in that the beta-glucanases are selected from the group consisting of those of the families GH5, GH6, GH7, GH12, GH16, GH17, GH74 or combinations thereof.

A malt-blotting method characterized in that it comprises incubating a malt comprising solution with an enzyme composition comprising the sequence arabinofuranosidase defined as SEQ ID NO: 1, the xylanase sequence defined as SEQ ID NO: 2 and beta-glucanases.

A method according to claim 3, wherein the beta-glucanases are selected from the group consisting of those of the families GH5, GH6, GH7, GH12, GH16, GH17, GH74 or combinations thereof.