WO2019109160A1 - Composition and method for malt mashing - Google Patents
Composition and method for malt mashing Download PDFInfo
- Publication number
- WO2019109160A1 WO2019109160A1 PCT/BR2018/050450 BR2018050450W WO2019109160A1 WO 2019109160 A1 WO2019109160 A1 WO 2019109160A1 BR 2018050450 W BR2018050450 W BR 2018050450W WO 2019109160 A1 WO2019109160 A1 WO 2019109160A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- malt
- enzymes
- addition
- enzyme
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C5/00—Other raw materials for the preparation of beer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C7/00—Preparation of wort
- C12C7/04—Preparation or treatment of the mash
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
Definitions
- 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.
- 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.
- 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.
- enzymatic preparations can be added which may contain ⁇ -amylases, ⁇ -glucanases, cellulases and hemicellulases to promote the extraction of carbohydrates independently of the enzymatic content present in the grain germinated.
- the product obtained, called wort is filtered.
- the addition of enzyme preparations also has the purpose of reducing their viscosity and facilitating filtration.
- corn grit as adjuvant, it is liquefied, that is, subjected to a specific enzymatic treatment with commercially available thermostable ⁇ -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.
- 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.
- 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.
- 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.), followeded 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the density values in degrees Plato (° P) In the second graph are the yield values, in%.
- the fourth graph are plotted the viscosity values in mPa.s.
- 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.
- the density values in degrees Plato (° P) In the second graph are the yield values, in%.
- 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.
- 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.
- 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.
- 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.
- 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.
- composition of the present invention allows, under the most drastic process conditions, a mean reduction in the concentration of beta-glucans greater than 50%.
- 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.
- 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.
- the malt solution which solution comprises water, malt and, optionally, pH and ionic strength correction agents
- 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).
- 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.
- 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.
- 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.
- arabinofuranosidase enzymes sequence defined as SEQ ID NO: 1
- xylanase of sequence defined as SEQ ID NO: 2
- 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.
- 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.
- 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 TM Color Gluca Test® kit.
- 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.
- 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.
- 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.
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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
COMPOSIÇÃO E MÉTODO PARA MOSTURAÇÃO DE MALTE COMPOSITION AND METHOD FOR MALT SHOWING
CAMPO DA INVENÇÃO FIELD OF THE INVENTION
[001] A presente invenção refere-se a uma composição voltada à hidrólise de polissacarideos do malte e ao método de mosturação do mesmo para a fabricação de bebidas. Mais especificamente, a presente invenção compreende uma composição enzimática definida e um método de mosturação de malte que envolve a incubação do mesmo com a referida composição. A presente invenção possui aplicação na indústria de alimentos, em especial, nas cervejarias. 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.
FUNDAMENTOS DA INVENÇÃO BACKGROUND OF THE INVENTION
[002] O processo industrial atual para a produção de cerveja baseia-se, essencialmente, nos mesmos princípios entre cervejarias. Os ingredientes básicos empregados na sua produção são água tratada, com pH e composição iônica corrigidos, malte, lúpulo, levedura do gênero Saccharomyces, além de, eventualmente, outras fontes de carboidratos chamadas de "adjuntos", que são empregadas em proporções diferentes e podem ser selecionadas dentre grítz de milho, arroz ou xarope de alta maltose, por exemplo. [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.
[003] No processo, o malte de cevada é, inicialmente, moído. Após adição de água e correção de pH e força iônica, tem-se o início da etapa de maceração, na qual o malte moído hidratado é submetido à agitação com aumento gradual de temperatura, em rampas com período de tempo definidos, permitindo a ação das diversas enzimas do próprio malte (endógenas) : b-glucanases (por volta de 45°C) ; proteases (por volta de 52°C); b-amilases (por volta de 60°C) , e - amilases (por volta de 70°C) . Em 77°C, estas enzimas endógenas são inativadas.
[004] Em função da grande variedade na qualidade do malte comercial, bem como de seu preço, são adicionadas preparações enzimáticas podendo conter a-amilases, b-glucanases , celulases e hemicelulases para favorecer a extração de carboidratos independentemente do conteúdo enzimático presente no grão germinado. Após a maceração, o produto obtido, chamado mosto, é filtrado. A adição de preparações enzimáticas também tem a finalidade de reduzir sua viscosidade e facilitar a filtragem. Na utilização de grítz de milho como adjunto, este é liquefeito, ou seja, submetido a um tratamento enzimático especifico com a-amilases termoestáveis comercialmente adquiridas para a degradação do amido, seu principal componente, em glicose. O adjunto liquefeito é, então, misturado ao mosto ainda na etapa de maceração . 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.
[005] Após a filtragem, o material insolúvel contendo malte não hidrolisado é descartado. Em seguida o mosto sofre a adição de lúpulo de amargor (mais rico em a-ácidos) e é submetido à fervura por 50 minutos. Mais lúpulo é adicionado, agora de aroma, e a fervura prossegue por mais 10 minutos. A fervura tem por objetivo a esterilização do liquido, a extração dos componentes do lúpulo, a eliminação de componentes voláteis indesejados e a redução de volume de água, quando necessário. Em seguida, o mosto repousa por 20- 30 minutos, para promover a separação do trub, mistura de proteínas precipitadas e outros componentes insolúveis que serão removidos em um decantador arrefecedor hidrodinâmico, também conhecido como Whirlpool ou rotapool. O mosto é, então, resfriado e oxigenado para, finalmente, receber as leveduras e iniciar-se o processo de fermentação. Nesta
etapa, podem ser adicionadas preparações contendo endopeptidase prolina-especifica e a-acetolactato descarboxilase, com o objetivo de controle de turbidez e de diacetil, respectivamente . A fermentação ocorre por 5-7 dias, em temperaturas baixas ( 12-14 °C, com Saccharomyces sp . ) , seguida por um período de maturação a 0°C por 3-7 dias, variável em função da concentração de diacetil encontrada após a fermentação. Em seguida à maturação, a cerveja é filtrada, carbonatada, diluída, sofre a adição de estabilizante de espuma, de antioxidante e, então, é envazada . 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.
[006] Um ponto que pode ser aprimorado no processo atual é o de aproveitamento do malte, reduzindo a quantidade de malte não hidrolisado por meio de composições enzimáticas exógenas mais eficientes. Neste sentido, WO09805788 descreve um processo para a produção de bebidas alcoólicas ao qual é adicionada uma mistura de enzimas, cuja composição compreende pelo menos o uso de uma endoxilanase, uma arabinofuranosidase, uma -amilase, uma endopeptidase, uma 1-3 ( 4 ) -b-glucanase e podendo conter ainda uma amilase sacarificante e/ou uma exopeptidase . W02015032850 comenta sobre a criação de um método para a redução da viscosidade do mosto pela adição de uma arabinofuranosidase da família GH43. WO2005118769 apresenta uma composição enzimática contendo uma endoglucanase de T. reesei e uma xilanase GH10 derivada de Aspergillus, que é útil na etapa de trituração e filtração do processo de fabricação da cervej a . EP3017706 descreve métodos e composições enzimáticas para a preparação de cereais maltados e sua utilização na fabricação de alimentos e bebidas, mencionando o uso de xilanases
combinadas com glucanases para a hidrólise do malte. 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] Embora existam várias composições propostas para a realização da mosturação do malte, a manutenção da atividade durante o maior período possível do processo é característica fundamental. Mesmo dentro de um grupo de enzimas que possuem a mesma atividade, existem diferenças de velocidade, de eficiência catalítica e de resistência às condições do processo de hidrólise. Ainda, nem todas as enzimas que possuem a mesma atividade catalítica apresentarão sinergia com as demais enzimas que constituem uma composição enzimática: em boa parte das associações, o efeito é nulo ou, até mesmo negativo, reduzindo a eficácia da composição como um todo. Levando-se em consideração a questão da rampa de temperatura convencionalmente empregada para a execução da etapa de mosturação do malte, é importante que a composição enzimática possa apresentar atividade dentro da faixa estipulada para garantir o máximo de eficiência do processo. [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.
[008] Portanto, carece-se de composições que possuam um conjunto enzimático capaz de fechar o ciclo da mosturação do malte com eficácia ampliada, mantendo sua atividade na temperatura do processo durante o maior período e com o mínimo de carga enzimática possíveis sem intervir no processo posterior de fermentação do mosto, dado que, conforme o conjunto de enzimas empregado na mosturação, compostos inibitórios podem ser liberados no meio de forma a reduzir a eficiência global da produção de cerveja ou de outras bebidas à base de fermentados de cereais. 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.
BREVE DESCRIÇÃO DA INVENÇÃO BRIEF DESCRIPTION OF THE INVENTION
[009] A presente invenção trata-se de uma composição
enzimática para a mosturação do malte e de um método de mosturação de malte com a referida composição. Mais especificamente, a presente invenção trata-se de uma composição enzimática que compreende a arabinofuranosidase de sequência definida como SEQ ID NO: 1, a xilanase de sequência definida como SEQ ID NO: 2 e beta-glucanases , além do método de mosturação de malte com a composição da presente invenção . 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.
[010] A presente invenção apresenta a vantagem de ser ativa nas condições processuais mais drásticas, permitindo um incremento de °Brix do mosto e, portanto, de rendimento de hidrólise, além de promover uma redução de sua viscosidade. Ainda, a presente invenção apresenta a vantagem de poder afetar positivamente a fermentação do mosto, proporcionando uma redução na quantidade de células mortas e, consequentemente, possibilitando um maior reuso do fermento empregado. 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.
BREVE DESCRIÇÃO DAS FIGURAS BRIEF DESCRIPTION OF THE DRAWINGS
[011] A Figura 1 mostra um gráfico comparativo de atuação das enzimas em sinergia buscando a melhor combinação com menor número de enzimas possível. Mixed E contém 9 enzimas: SEQ ID NO: 1, Gluc_GH16, b-GlucjGHl, oí-Xyl_GH31, b-Cg1_OH39, Exo_Xeg_GH5, AraXyl_GH5, Man_GH5 e SEQ ID NO: 2. Mixed O contém 6 enzimas: as anteriores menos -Xyl_GH31, Exo_Xeg_GH5 e Man_GH5. Mixed U contém 5 enzimas: as anteriores menos Gluc_GH16. Mixed Y contém 4 enzimas: as anteriores menos SEQ ID NO: 1. Mixed T contém 5 enzimas: SEQ ID NO: 1, Gluc_GHl 6 , b-Cg1_OH39, AraXyl_GH5 e SEQ ID NO: 2. Mixed Z contém 4 enzimas: as anteriores menos Gluc_GH16. Mixed AB contém 3 enzimas: as anteriores menos b-Cg!_OH39.
Mixed AE contém 4 enzimas: SEQ ID NO: 1, Gluc_GH16, b- Xyl_GH39 e SEQ ID NO: 2. Mixed AF contém 3 enzimas: as anteriores menos b-Cg1_OH39. Mixed AA contém SEQ ID NO: 1, b-Cg1_OH39 e SEQ ID NO: 2. Mixed AC contém apenas 2 enzimas: SEQ ID NO: 1 e SEQ ID NO: 2. Ao lado de cada combinação foi plotado o seu respectivo controle indicando a contribuição, em atividade, que cada enzima apresentaria caso estivesse atuando isoladamente. Os valores das reações sinérgicas apresentados são a média e o desvio padrão. 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.
[012] A Figura 2 mostra um gráfico com as curvas de concentração das enzimas arabinofuranosidase SEQ ID NO: 1 e xilanase SEQ ID NO: 2 atuando em sinergia sobre bagaço de malte com gritz proveniente de um processo com adição da enzima Laminex® Super 3G e sobre o bagaço de malte com xarope de alta maltose proveniente de um processo com adição da enzima comercial Bioglucanase® GB. Os experimentos foram realizados em rampas de temperatura otimizadas. Os valores representam a média e o desvio padrão. 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.
[013] Na Figura 3 são apresentados os resultados dos parâmetros avaliados no "Mosto Congress" para teste das enzimas SEQ ID NO: 1 e SEQ ID NO: 2 com malte e adição da enzima comercial Bioglucanase® GB. No primeiro gráfico estão plotados os valores de densidade em graus Plato (°P) . No segundo gráfico estão os valores de rendimento, em %. No terceiro gráfico estão os valores de concentração de b- glucanos remanescentes no mosto, em mg/L. No quarto gráfico estão plotados os valores de viscosidade em mPa.s. Branco : malte com adição de Bioglucanase® GB; SEQ ID NO: 2: reação com adição de 0,6 mg/mL de SEQ ID NO: 2; SEQ ID NO: 1 : reação com adição de 0,6 mg/mL de SEQ ID NO: 1; SEQ ID NO: 1 + 2 :
reação com adição de SEQ ID NO: 1 e SEQ ID NO: 2 ou 3 a 0, 6 mg/mL cada. A enzima comercial Bioglucanase® GB foi adicionada em todas as condições na dosagem padrão de 150 g/tonelada de malte. Os valores representam a média e o desvio padrão. 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] A Figura 4 mostra os resultados obtidos para os parâmetros avaliados no "Mosto Congress" para teste das enzimas SEQ ID NO: 1 e SEQ ID NO: 2 com malte e adição da enzima comercial Rohalase® Barley L. No primeiro gráfico estão plotados os valores de densidade em graus Plato (°P) . No segundo gráfico estão os valores de rendimento, em %. No terceiro gráfico estão os valores de concentração de b- glucanos remanescentes no mosto, em mg/L. No quarto gráfico estão plotados os valores de viscosidade em mPa.s. Malte puro : malte apenas, sem adição de qualquer enzima; SEQ ID NO: 2: reação com adição de 0,6 mg/mL de SEQ ID NO: 2; SEQ ID NO: 1 : reação com adição de 0,6 mg/mL de SEQ ID NO: 1; SEQ ID NO: 1 + 2 : reação com adição de SEQ ID NO: 1 e SEQ ID NO: 2 a 0, 6 mg/mL cada. A enzima comercial Rohalase® Barley L foi adicionada em todas as condições, exceto no malte puro, na dosagem padrão de 75 g/tonelada de malte. Os valores representam a média e o desvio padrão. [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.
[015] A Figura 5 ilustra os resultados dos parâmetros avaliados no "Mosto Congress" no teste das enzimas SEQ ID NO: 1 e SEQ ID NO: 2 com malte e adição da enzima comercial Laminex® MaxFlow 4G. No primeiro gráfico estão plotados os valores de densidade em graus Plato (°P) . No segundo gráfico estão os valores de rendimento, em %. No terceiro gráfico estão os valores de concentração de b-glucanos remanescentes no mosto, em mg/L. No quarto gráfico estão plotados os
valores de viscosidade em mPa.s. Malte puro : malte apenas, sem adição de qualquer enzima; SEQ ID NO: 2: reação com adição de 0,6 mg/mL de SEQ ID NO: 2; SEQ ID NO: 1 : reação com adição de 0,6 mg/mL de SEQ ID NO: 1; SEQ ID NO: 1 + 2: reação com adição de SEQ ID NO: 1 e SEQ ID NO: 2 a 0, 6 mg/mL cada. A enzima comercial Laminex® MaxFlow 4G foi adicionada em todas as condições, exceto no malte puro, na dosagem padrão de 75 g/tonelada de malte. Os valores apresentados representam a média e o desvio padrão. 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.
[016] A Figura 6 apresenta um comparativo de arabinofuranosidase SEQ ID NO: 1 (indicada como Abf) e xilanase SEQ ID NO: 2 (indicada como Xyl) adicionadas de b- glucanases comerciais. Todos os parâmetros avaliados foram plotados em porcentagem, sendo que os valores das enzimas comerciais foram considerados como 100%. Bioglucanase, Rohalase, laminex : adição apenas de cada uma dessas enzimas comerciais em sua dosagem padrão. B+Xyl+Abf: adição da enzima comercial Bioglucanase, de SEQ ID NO: 1 e SEQ ID NO: 2, ambas as SEQ a 0,6 mg/mL; R+Xyl+Abf: adição da enzima comercial Rohalase® Barley L e das enzimas SEQ ID NO: 1 e SEQ ID NO: 2; I+Xyl+Abf: adição da enzima comercial Laminex® MaxFlow 4G e das enzimas SEQ ID NO: 1 e SEQ ID NO: 2. Os valores representam a média e o desvio padrão. 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.
[017] A Figura 7 mostra os resultados do teste no "Mosto Congress" para verificar o efeito da concentração das enzimas comerciais, de SEQ ID NO: 1 e SEQ ID NO: 2 sobre o malte. As enzimas comerciais Bioglucanase® GB, Rohalase® Barley L e Laminex® MaxFlow 4G foram testadas na concentração de 1,2 mg/mL e comparadas com teste anterior na dosagem padrão. Malte puro : malte apenas, sem adição de qualquer enzima;
Bioglucanase + SEQ ID NO: 1 + 2: enzima comercial Bioglucanase® GB na dosagem padrão (150 g/tonelada de malte) mais adição de SEQ ID NO: 1 + SEQ ID NO: 2, ambas a 0,6 mg/mL. Os valores representam a média e o desvio padrão. 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.
[018] A Figura 9 mostra os resultados do teste de avaliação no "Mosto Congress" das enzimas SEQ ID NO: 1 e SEQ ID NO: 2 como substituintes das b-glucanases comerciais. Malte puro : malte apenas, sem adição de qualquer enzima; Bioglucanase (150 g/ton) : reação com adição apenas da enzima comercial Bioglucanase® GB na dosagem padrão de 150 g/tonelada de malte; SEQ ID NO: 1 + 2 : reação com adição apenas de SEQ ID NOs : 1 e 2 (ambas as enzimas a 0, 6 mg/mL) ; Bioglucanase (150 g/ton) + SEQ ID NO: 1 + 2 : reação com adição da enzima comercial Bioglucanase® GB na dosagem padrão de 150 g/tonelada de malte mais SEQ ID Nos 1 e 2 (a 0, 6 mg/mL) . Os valores são médias ± o desvio padrão. 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.
DESCRIÇÃO DETALHADA DA INVENÇÃO DETAILED DESCRIPTION OF THE INVENTION
[019] A presente invenção trata-se de uma composição enzimática que compreende a arabinofuranosidase de sequência definida como SEQ ID NO: 1, a xilanase de sequência definida como SEQ ID NO: 2 e beta-glucanases . 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.
[020] Entende-se por arabinofuranosidase como sendo uma enzima que atua sobre extremidades não redutoras de arabinooligossacarideos e cadeias laterais de polissacarideos que contenham resíduos de arabinose unidos por ligações dos tipos CÍ-1,2, -1,3 e/ou -1,5. 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] Entende-se por xilanase como sendo a enzima responsável pela clivagem das ligações do tipo b-1,4 entre os resíduos de xilose na cadeia principal de xilanos e heteroxilanos .
[022] Entende-se por beta-glucanases como sendo as enzimas responsáveis pela clivagem das ligações dos tipos b- 1,3 e b-1,4 entre os resíduos de glicose em glucanos. As beta-glucanases da composição do presente invento podem ser selecionadas dentre o grupo que compreende as famílias das hidrolases glicosídicas GH5, GH6, GH7, GH12, GH16, GH17, GH74 ou combinações entre estas. As beta-glucanases da composição da presente invenção podem ser provenientes de bactérias ou fungos, porém não se restringindo a estes, sendo estas beta-glucanases de origem natural ou heteróloga, com ou sem mutações, provenientes ou não de mutação dirigida. [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] Comparativamente a três composições base compostas por beta-glucanases, a composição da presente invenção permite, nas condições mais drásticas de processo, uma redução média da concentração de beta-glucanos superior a 50%. [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] A composição pode ser apresentada sob a forma de solução ou como um sólido. Neste último caso, a composição pode se apresentar tanto sob a forma de pó liofilizado ou como grânulos. É opcional a adição de um estabilizante e/ou um conservante à composição. [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.
[025] A presente invenção refere-se, ainda a um método de mosturação de malte caracterizado pelo fato de incubar a solução compreendendo malte com uma composição enzimática que compreende a arabinofuranosidase de sequência definida como SEQ ID NO: 1, a xilanase de sequência definida como SEQ ID NO: 2 e beta-glucanases. 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] Novamente, as beta-glucanases da composição da etapa (c) podem ser selecionadas dentre o grupo que compreende as famílias das hidrolases glicosídicas GH5, GH6,
GH7, GH12, GH16, GH17, GH74 ou combinações entre estas. Ainda, as beta-glucanases da composição da etapa (c) podem ser provenientes de bactérias ou fungos, porém não se restringindo a estes, sendo estas beta-glucanases de origem natural ou heteróloga, com ou sem mutações, provenientes ou não de mutação dirigida. [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 .
[027] A incubação a que se refere o método da presente invenção pode incluir a submissão da solução de malte, solução esta que compreende água, malte e, eventualmente, agentes de correção do pH e da força iônica, à agitação com aumento gradual de temperatura, em faixas e rampas com período de tempo definidos conforme as práticas já conhecidas no estado da técnica. 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] Exemplo : O bagaço de malte é o nome dado ao material remanescente não hidrolisado da mostura do malte. Este é rico em celulose (17%) e, principalmente, hemiceluloses (39%), especialmente arabinoxilanos (Valverde, 1994; Bartolomé et ai, 2002; Mandalari et ai., 2005; Mussato et ai., 2006; Robertson et ai., 2010) . [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).
[029] A partir desta informação foram selecionadas 22 enzimas recombinantes , dentre celulases e hemicelulases , com potencial aplicação no processo de mostura do malte para compor um coquetel enzimático personalizado que permitisse um maior fracionamento de arabinoxilanos e glucanos e, consequentemente, um aumento na produtividade e na redução de custos das cervejarias. 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.
[030] Na Tabela 1 estão descritas as enzimas recombinantes que foram testadas e a família das hidrolases glicosídicas a qual pertencem. The recombinant enzymes that have been tested and the family of the glycosidic hydrolases to which they belong are described in Table 1.
[031] Estas 22 enzimas recombinantes foram expressas de
forma heteróloga em E. coli, purificadas por cromatografia de afinidade em resina de níquel (Ni-NTA Agarose - Qiagen) e avaliadas em reações de hidrólise contra o bagaço de malte com grítz ou xarope de alta maltose sob as condições de mostura normalmente empregadas pelas indústrias cervej eiras. [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] Tabela 1: enzimas recombinantes testadas [032] Table 1: recombinant enzymes tested
[033] Estes experimentos de hidrólise enzimática foram realizados em microtubos de 1,5 mL contendo 55 mg de bagaço de malte, 125 pL de água destilada, 225 pL de tampão acetato de sódio 250 mM pH 5,5 e 150 pL de enzima a ser testada. As reações de hidrólise foram incubadas em agitador com controle de temperatura, sendo a agitação de 700 rpm e o aumento da temperatura realizado de forma gradual de acordo com as rampas de mostura utilizadas nas cervejarias. Nestes testes foram utilizadas duas rampas de temperatura, uma chamada de padrão que iniciava a 45°C e a outra denominada de otimizada, que começava a 60°C. Ambas as rampas de temperatura se encerravam a 77°C. Ao final do processo, as amostras foram centrifugadas por 2 minutos a 14.000 x g para a sedimentação do material particulado. Os sobrenadantes obtidos foram utilizados para a quantificação de açúcar redutor liberado nas reações de hidrólise através do método colorimétrico com o reagente ácido 3 , 5-dinitrosalicílico - DNS (Miller, 1959) . 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).
[034] Das 22 enzimas testadas sob os bagaços de malte nas duas rampas de temperatura, foi selecionada pelo menos uma enzima representante de cada tipo de atividade para serem avaliadas em experimentos de sinergia. Esses testes tiveram por objetivo encontrar a melhor combinação com o menor número possível de enzimas para compor um coquetel enzimático.
[035] Nos experimentos de sinergia as reações de hidrólise foram preparadas como descrito anteriormente, com a única diferença de que as enzimas utilizadas foram diluídas 2,5 vezes quando comparadas com os testes de atividade realizados para cada uma delas isoladamente. Também foram preparadas reações controle negativo, ou seja, sem a adição de nenhuma enzima. Em cada combinação enzimática realizada foi sendo excluída uma atividade enzimática por vez, com intuito de se obter uma mistura com uma quantidade mínima de enzimas que apresentasse um ganho considerável na hidrólise de bagaço. A quantificação de açúcar redutor produzido nas reações foi determinada pelo método do reagente 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] Na Figura 1 são apresentados os resultados obtidos nos experimentos de combinação das enzimas durante os testes de sinergia. O valor de absorbância obtido nas reações de hidrólise enzimática foi subtraído do valor de absorbância dos respectivos brancos (reações realizadas nas mesmas condições, porém sem adição de enzimas) . [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).
[037] Verificou-se que com 9 enzimas atuando em sinergia {mixed E - SEQ ID NO: 1 (AbfD3), uma endo-glucanase, uma b- glucosidase, uma a-xilosidase , uma b-xilosidase, uma xiloglucanase, uma arabinoxilanase, uma mananase e SEQ ID NO: 2 (XyllOB) obtinha-se um valor médio de 11,3 unidades de absorbância (u.a.) . Reduzindo-se para 6 enzimas (mixed O - SEQ ID NO: 1, uma endo-glucanase, uma b-glucosidase, uma b- xilosidase, uma arabinoxilanase e SEQ ID NO: 2) a atividade total foi para 10,7 u.a. Com 5 enzimas (mixed T - SEQ ID NO: 1, uma endo-glucanase, uma b-xilosidase, uma arabinoxilanase e SEQ ID NO: 2) obteve-se um valor médio de 9,9 u.a. e,
considerando o desvio padrão, esta redução na atividade de 9 para 5 enzimas não foi significativamente relevante. Com 3 enzimas {mixed AF - SEQ ID NO: 1, uma endo-glucanase e SEQ ID NO: 2) a atividade média foi de 8,5 u.a., enquanto que com apenas 2 enzimas (mixed AC - SEQ ID NO: 1 e SEQ ID NO: 2) a atividade foi de 7,4 u.a. e, novamente, considerando o desvio padrão entre 5, 3 e 2 enzimas essa variação na atividade média não foi significativa. Desta forma, a melhor relação custo x beneficio, com relação ao nivel de material remanescente não hidrolisado no bagaço de e quantidade de enzimas necessárias para tal hidrólise, foi com apenas 2 enzimas, a arabinofuranosidase SEQ ID NO: 1 e a xilanase SEQ ID NO: 2. 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] Foi ainda realizada uma avaliação em microescala de concentração das enzimas selecionadas, com o intuito de se obter a melhor concentração de uso da arabinofuranosidase e da xilanase no coquetel enzimático. Nesta avaliação foram preparadas reações de hidrólise enzimática usando a enzima arabinofuranosidase SEQ ID NO: 1 em uma concentração fixa de 0,6 mg/mL e variações da concentração da xilanase SEQ ID NO: 2, entre 0 a 1,2 mg/mL. Essas hidrólises foram preparadas como descrito anteriormente usando bagaço com grítz e bagaço com xarope de alta maltose sob as condições de rampa otimizada de temperatura. [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] Na Figura 2 são apresentados os resultados desta avaliação. Foi verificado que em concentrações acima de 0,6 mg/mL da xilanase não são obtidos ganhos significativos de liberação de açúcares. Observou-se ainda que o comportamento das duas enzimas em sinergia foi bem semelhante contra os dois tipos de bagaços testados, sendo estes, inclusive,
provenientes de processos de produção com adição de diferentes enzimas comerciais, indicando que a atuação dessas enzimas foi eficaz mesmo com variações no tipo de bagaço utilizado. [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] Posteriormente, as enzimas arabinofuranosidase, de sequência definida como SEQ ID NO: 1, e xilanase, de sequência definida como SEQ ID NO: 2, foram expressas em larga escala e purificadas para serem utilizadas em testes de mostura com o malte no equipamento "Mosto Congress" (Banho de Brassagem - Fluxo Tecnologia) . Este equipamento permite a análise de processamento do mosto, possibilitando reproduzir desde a moagem do malte misturado com água aquecida até etapa de maceração e simula a rampa de temperatura . [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] As reações de hidrólise realizadas nos micro reatores do equipamento "Mosto Congress" foram preparadas usando um volume final de 100 mL . Estas reações continham 12,5 g do malte recém-moido em moinho de discos (Bílhler) , CaCÍ2 até se ter a concentração final de 0,3 mg/mL e a enzima comercial Bioglucanase® GB na dosagem padrão recomendada pelo fabricante (150 g/tonelada de malte), neste caso foi usado 1,8 pL da enzima. Foi ainda adicionado à mistura reacional as quantidades necessárias das enzimas SEQ ID NO:l e 2 até se obter a concentração de 1,2 mg/mL (0,6 mg/mL de xilanase + 0,6 mg/mL de arabinofuranosidase) e água destilada até completar o volume de reação de 100 mL . Também foram preparadas reações controle sem adição de nenhuma enzima, assim como reações contendo apenas a enzima Bioglucanase® GB, reações contendo a enzima Bioglucanase® GB com a adição da xilanase SEQ ID NO: 2 na concentração de 0,6 mg/mL e a
enzima Bioglucanase® GB com a arabinofuranosidase SEQ ID NO: 1 também na concentração de 0,6 mg/mL. Estas reações de hidrólise foram realizadas sob agitação de 100 rpm e utilizando as condições de rampa otimizada de temperatura. [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.
[042] Após finalizada a rampa de temperatura, as reações foram mantidas a temperatura ambiente, os volumes acertados de acordo com os respectivos valores iniciais (para correção do volume de água evaporado durante o aumento de temperatura) e o resultado avaliado imediatamente. Os parâmetros avaliados foram o tempo de filtração, concentração de b- glucanos, densitometria e viscosidade. O tempo de filtração consiste na contagem manual do tempo necessário para que o material particulado se separe do liquido, através de um filtro de papel, com observação visual do operador. A concentração de b-glucanos é medida em espectrofotômetro, no comprimento de onda de 550 nm, em cubetas plásticas de 3 mL . Para tal, o liquido filtrado proveniente da mosturação é incubado por 30 minutos com a solução A do kit Enzitec™ Color Gluca Test®. Como controle negativo, essa solução é incubada com igual volume de água. Os valores obtidos de concentração de b-glucanos são em mg/L, tendo como referencial uma curva padrão. Os dados de tempo de filtração e concentração de b- glucanos podem ser utilizados para calcular a eficiência de uma determinada enzima adicionada no processo. 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] Os graus Brix ou graus Plato foram medidos em densímetro (Anton Paar DMA 4.500 Density Meter) . Essas medidas foram realizadas a 20°C com aproximadamente 50 mL de amostra (líquido filtrado proveniente da mosturação), sendo que as duas primeiras medidas são feitas com água e as leituras são sequenciais e automáticas.
[044] O último parâmetro avaliado foi a viscosidade das amostras, medidas em um viscosimetro (Brooksfield Viscometer DV-I Prime) , sendo as leituras realizadas a 20 °C durante 10 minutos, gerando um valor médio em mPa.s. [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] Este mesmo teste de hidrólise do malte no equipamento "Mosto Congress" foi realizado com outros coquetéis enzimáticos comerciais que também apresentam atividade declarada de b-glucanases . Ao invés da Bioglucanase® GB foram utilizados as enzimas Rohalase® Barley L (AB Enzymes) e Laminex® MaxFlow 4G (Danisco) . Estas duas enzimas foram utilizadas na dosagem padrão recomendada pelo fabricante de 75 g/tonelada de malte. [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.
[046] Como pode ser observado nas Figuras 3, 4, 5 e 6, o uso das das enzimas SEQ ID NO : 1 e 2 em combinação com diferentes b-glucanases comerciais promoveu o aumento no valor Brix e no rendimento da hidrólise do malte, bem como reduziu a concentração de b-glucanos remanescentes no mosto e também diminuiu a sua viscosidade. O ganho médio com a composição do presente invento para o Brix foi de 3,9% e, para o rendimento, 4,43%. A redução média na concentração de b-glucanos foi de 55,92% e, na viscosidade, de 2,72%. 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] Também foram realizados experimentos para avaliar se o aumento na dosagem utilizada das enzimas comerciais Bioglucanase® GB, Rohalase® Barley L e Laminex® MaxFlow 4G provocaria um resultado semelhante ao obtido com a adição do das duas enzimas na hidrólise enzimática do malte. Nestes testes, foram preparadas reações de hidrólise no equipamento "Mosto Congress" usando as enzimas comerciais na mesma concentração final das demais, ou seja, de 1,2 mg/mL nas reações ao invés das concentrações recomendadas pelos
fornecedores. Para tal, a concentração das enzimas comerciais foi determinada através de leitura da absorbância em 280 nm no equipamento NanoDrop 2000c da Thermo Scientific™. O mesmo padrão dos testes anteriores com relação a rampa de temperatura, volume de reação e padrão de agitação foram adotados nesta avaliação. [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] Como pode ser verificado na Figura 7, o aumento na concentração das três enzimas comerciais não foi eficaz. Este resultado indica que as duas enzimas SEQ ID NO: 1 e SEQ ID NO: 2 têm um papel complementar na hidrólise do malte, atuando sobre alvos diferentes daqueles das enzimas comerciais, de tal forma que uma hidrólise mais eficiente independe do aumento na concentração das b-glucanases comerciais testadas. [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.
[049] Outra análise feita foi com relação ao papel desempenhado pela adição das enzimas da composição do presente invento sobre o malte no processo de mosturação, ou seja, se as duas enzimas da referida composição estariam atuando com funções complementares ou substituintes às enzimas comerciais. Para este teste, foram realizadas hidrólises usando SEQ ID NO: 1 e SEQ ID NO: 2 a 0, 6 mg/mL na presença ou não de Bioglucanase® GB, na dosagem padrão de 150 g/tonelada de malte, seguindo o mesmo padrão dos testes anteriores realizados no equipamento "Mosto Congress". 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.
[050] Os resultados obtidos neste teste, apresentados na Figura 8, demonstram que SEQ ID NO: 1 e SEQ ID NO: 2 têm um efeito de complementação de atividade da preparação enzimática comercial contendo b-glucanases mais pronunciado do que como substituinte da mesma no processo de mosturação.
[051] Testes de microfermentação do mosto obtido a partir da mostura do malte com as enzimas arabinofuranosidase SEQ ID NO: 1, xilanase SEQ ID NO: 2 e beta-glucanases foram realizados. Para este teste, a mostura foi realizada como descrito anteriormente no equipamento "Mosto Congress", porém utilizando um volume de 400 mL em cada copo. As reações controle continham apenas malte, sem adição de qualquer enzima; nas reações branco BG foi adicionada apenas a enzima comercial Bioglucanase® GB na dosagem padrão de 150 g/tonelada de malte, enquanto nas reações de interesse houve adição, além da enzima comercial Bioglucanase® GB, de 0, 6 mg/mL de cada enzima purificada, mantendo a estequiometria de 1:1 entre SEQ ID NO: 1 e SEQ ID NO: 2. A mosturação foi realizada na rampa de temperatura otimizada. Após finalizada a rampa de temperatura, as reações foram mantidas a temperatura ambiente, os volumes acertados de acordo com os respectivos valores iniciais e o primeiro parâmetro avaliado foi o tempo de filtração, seguido pela determinação da concentração de b-glucanos, densitometria e viscosidade. 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] Após avaliação da etapa de mosturação, uma parte do mosto filtrado (200 mL) foi incubada com 15 gramas de fermento, sob agitação de 148 rpm a temperatura ambiente por 24 horas. O material resultante foi filtrado, em filtro de papel, para separação do fermento e analisado no sistema Alcolyzer Beer (Anton Paar) . Este sistema de análise fornece os valores de extrato original (em °P) e extrato aparente (em %), densidade em g/cm3, teor alcoólico em %, quantidade de células mortas, vitalidade e um valor do grau de fermentação ocorrido (ADF Apparent Degree of Fermentation) .
[053] A Tabela 2 apresenta os resultados obtidos no teste de microfermentação . Os valores representam a médias e o desvio padrão. Malte refere-se ao mosto proveniente de uma mosturação contendo apenas malte, sem adição de qualquer enzima; Branco BG refere-se ao mosto proveniente de mosturação com adição da preparação enzimática comercial Bioglucanase® GB na dosagem padrão (150 g/tonelada de malte) ; BG + SEQ ID NO: 1 e SEQ ID NO: 2 (1:1) = adição da[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 na dosagem padrão e das enzimas SEQ ID NO: 2 e SEQ ID NO: 1, ambas a 0, 6 mg/mL. 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] Tabela 2: resultados de microfermentação [054] Table 2: microfermentation results
[055] A adição das enzimas SEQ ID NO: 1 e SEQ ID NO: 2 levou a um considerável aumento nos valores de extrato aparente e original e, além disso, resultou em uma redução de 55,25% na quantidade de células mortas em relação à mosturação sem adição de enzimas (apenas malte), permitindo assim mais ciclos de reutilização do fermento adicionado.
[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
1. Composição para mosturação de malte caracterizada por compreender a arabinofuranosidase de sequência definida como SEQ ID NO: 1, a xilanase de sequência definida como SEQ ID NO: 2 e beta-glucanases . 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.
2. Composição, de acordo com a reivindicação 1, caracterizada por as beta-glucanases serem selecionadas dentre o grupo que compreende as das famílias GH5, GH6, GH7, GH12, GH16, GH17, GH74 ou combinações entre estas. 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.
3. Método para mosturação de malte caracterizado por compreender a incubação de solução compreendendo malte com uma composição enzimática que compreende a arabinofuranosidase de sequência definida como SEQ ID NO: 1, a xilanase de sequência definida como SEQ ID NO: 2 e beta- glucanases . 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.
4. Método, de acordo com a reivindicação 3, caracterizado por as beta-glucanases serem selecionadas dentre o grupo que compreende as das famílias GH5, GH6, GH7, GH12, GH16, GH17, GH74 ou combinações entre estas.
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.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BR102017026595-1A BR102017026595A2 (en) | 2017-12-08 | 2017-12-08 | COMPOSITION AND METHOD FOR MALT SHOWING |
BRBR1020170265951 | 2017-12-08 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2019109160A1 true WO2019109160A1 (en) | 2019-06-13 |
Family
ID=66749980
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/BR2018/050450 WO2019109160A1 (en) | 2017-12-08 | 2018-12-06 | Composition and method for malt mashing |
Country Status (2)
Country | Link |
---|---|
BR (1) | BR102017026595A2 (en) |
WO (1) | WO2019109160A1 (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015032850A1 (en) * | 2013-09-05 | 2015-03-12 | Novozymes A/S | Method for production of brewers wort |
EP3017706A1 (en) * | 2014-11-05 | 2016-05-11 | Dupont Nutrition Biosciences ApS | Enzymes for malting |
-
2017
- 2017-12-08 BR BR102017026595-1A patent/BR102017026595A2/en unknown
-
2018
- 2018-12-06 WO PCT/BR2018/050450 patent/WO2019109160A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015032850A1 (en) * | 2013-09-05 | 2015-03-12 | Novozymes A/S | Method for production of brewers wort |
EP3017706A1 (en) * | 2014-11-05 | 2016-05-11 | Dupont Nutrition Biosciences ApS | Enzymes for malting |
Non-Patent Citations (5)
Title |
---|
GOLDBECK, R. ET AL.: "Development of hemicellulolytic enzyme mixtures for plant biomass deconstruction on target biotechnological applications", APPL MICROBIOL BIOTECHNOL, vol. 98, 2014, pages 8513 - 8525, XP035405929, doi:10.1007/s00253-014-5946-6 * |
GONÇALVGAMMAS, T. A. ET AL.: "Functional characterization and synergic action of fungal xylanase and arabinofuranosidase for production of xylooligosaccharides", BIORESOUR TECHNOL., vol. 119, 2012, pages 293 - 299, XP028449077, doi:10.1016/j.biortech.2012.05.062 * |
MACHADO, C. B. ET AL.: "Increased biomass saccharification by supplementation of a commercial enzyme cocktail with endo- arabinanase from Bacillus licheniformis", BIOTECHNOL LETT., vol. 37, no. 7, July 2015 (2015-07-01), pages 1455 - 1462, XP055616440 * |
SORENSEN, H. R. ET AL.: "Enzymatic hydrolysis of wheat arabinoxylan by a recombinant ''minimal'' enzyme cocktail containing beta-xylosidase and novel endo-1,4-beta-xylanase and alpha-l-arabinofuranosidase activities", BIOTECHNOL PROG., vol. 23, no. 1, 2007, pages 100 - 107, XP002520699, doi:10.1021/BP0601701 * |
SORENSEN, H. R. ET AL.: "Synergistic enzyme mechanisms and effects of sequential enzyme additions on degradation of water insoluble wheat arabinoxylan", ENZYME AND MICROBIAL TECHNOLOGY, vol. 40, no. 4, 2007, pages 908 - 918, XP005892515, doi:10.1016/j.enzmictec.2006.07.026 * |
Also Published As
Publication number | Publication date |
---|---|
BR102017026595A2 (en) | 2019-06-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Steiner et al. | Comparison of beer quality attributes between beers brewed with 100% barley malt and 100% barley raw material | |
CA2348163C (en) | Preparation of wort and beer of high nutritional value, and corresponding products | |
AU2012307299B2 (en) | Compositions comprising enzymes with endo - 1, 4 - beta - xylanase activity and enzymes with endo - 1, 3 (4) - beta glucanase activity | |
EP3225634A1 (en) | Xylanase enzyme activity | |
EP1812547A1 (en) | Mashing process and enzyme composition useful therein | |
Mezgebe et al. | Relationship between waxy (high amylopectin) and high protein digestibility traits in sorghum and malting quality | |
CA2268506A1 (en) | Gluten-free beer containing rice malt | |
Li et al. | Beers | |
WO2019109160A1 (en) | Composition and method for malt mashing | |
JP7152859B2 (en) | Method for producing rooting-inhibited malt | |
WO2019219601A2 (en) | Method for production of brewers wort | |
US20020012718A1 (en) | Gluten-free beer | |
Guo et al. | Effect of kilning on the composition of protein and arabinoxylan in wheat malt | |
WO1999049011A1 (en) | Application of lipase in brewing | |
US1737279A (en) | Cereal extract and process of making same | |
KR102567366B1 (en) | Saccharide composition for fermented alcoholic beverage and its manufacturing method by using of the same | |
CN106636037A (en) | Compound enzyme preparation as well as preparation method and application thereof | |
Makuru | Evaluation of recombinant yeast strains expressing a xylanase, amylase or an endo-glucanase in brewing | |
Owuama et al. | Effect of Discorea cayensis and Ipomoea batatas extracts on sugar content of sorghum mash | |
CN107881044A (en) | A kind of method for promoting barley germination during beer brewing using alga oligosaccharides | |
Sim | The enzymomology of the malting, milling, mashing and fermentation processes within the Scotch malt whisky industry | |
EA020607B1 (en) | Process for preparing oatmeal beverage |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 18886671 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 18886671 Country of ref document: EP Kind code of ref document: A1 |