Improved production of rye-based alcoholic beverages

Abstract

The present invention relates to the use of enzymes to produce rye-based spirits. More particularly, the present invention relates to a method for starch saccharification of ground rye grains using a xylanase having a reduced sensitivity to a rye XIP inhibitor.

Description

TECHNICAL FIELD

[0001] The present invention relates to distilling rye whiskey. More particularly, the present invention relates to enzymes that improve rye distillation yields.

[0002] INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] The content of the electronic submission of the sequence listing in the text file named“NB42166_SequenceListing.xml” was created on January 31, 2023, and is 16 KB in size, which is hereby incorporated by reference in its entirety. BACKGROUND

[0004] Rye-based whiskeys are known for their spicy, strong flavors. Rye whiskey was very popular in the United States before the enactment of Prohibition. Rye is often mixed with classic cocktails, such as the Manhattan cocktail and the Old Fashioned, for human consumption because of its robust flavor.

[0005] After Prohibition ended, the popularity of rye whiskey dropped significantly. Rye whiskey was considered a“low-end” whiskey. In contrast, the popularity of bourbon whiskey (distilled from corn mash) rose significantly after Prohibition, particularly because corn production was subsidized by the U.S. federal government, and farmers were incentivized to grow corn. The drop in popularity of rye whiskey continued through the 1980s.

[0006] However, starting in 2006, there was a resurgence of interest in classic cocktails, which spurred a resurgence of rye whiskey. Since then, rye whiskey production has grown by about 1200% in 2009.

[0007] Despite advances in fermenting and distilling grains, such as barley, corn, and rice (including the introduction of enzymes), rye fermentation and distillation remain challenging and expensive. Rye distilleries have low profit margins because rye is one of the most difficult grains to process. In fact, distilling with rye grain presents unique difficulties for distilleries.

[0008] Compared to other grains used for ethanol production, rye has a relatively low starch content. Starch is converted to fermentable sugars, which are in turn converted to ethanol by yeast during fermentation. Less starch means less ethanol, and therefore, less whiskey produced per weight unit of rye grain. Additionally, the starch in rye grain is also more difficult to access than in other grains. Rye is notoriously difficult to process.

[0009] Rye mashes have high water binding capacity and tend to form gels of high viscosity and stickiness that tend to cement to fermentation and distillation equipment. Gelled rye mashes are difficult to pump in industrial distillation facilities. High viscosity increases fouling on column plates, reducing heat transfer efficiency and leading to material loss. Ethanol recovery is reduced, and efficiency is also reduced. Rye mashes also tend to clog heat exchangers. Dryer capacity is reduced, and the risk of dryer internal fires is increased.

[0010] During fermentation, viscous rye mashes tend to foam. Excessive foaming leads to material spillage and loss, or requires the use of undesirably small volume fermenters.

[0011] The need for methods for rye fermentation and distillation continues. SUMMARY

[0012] According to an aspect of the invention, there is presented a method for producing rye whiskey, the method having the steps of: (a) providing a grain mash having at least 51 w / w% of rye kernels; (b) adding water to the grain mash to provide a mash; (c) pre-liquefying the mash of step (b); (d) gelatinizing the mash of step (c); (e) liquefying the mash of step (d) in the presence of a xylanase having reduced sensitivity to rye XIP inhibitors; (f) saccharifying the mash of step (e); (g) fermenting the mash of step (f) to produce a fermentation containing ethanol; (h) distilling the fermentation of step (g) to provide rye whiskey. Optionally, the xylanase is inhibited by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% by rye XIP inhibitors. Optionally, the xylanase is inhibited by less than 20% by rye XIP inhibitors.

[0013] Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 3.

[0014] Optionally, the rye kernels are malted. In other preferred embodiments, the rye kernels are unmalted. Optionally, the rye kernels are milled. Preferably, the grain comminution is 60, 70, 80, 85, 90, 95, 99, or 100 w / w% rye. Optionally, the grain comminution further comprises malted or unmalted barley, wheat, corn, rye, rice, cassava, oat flakes, or sorghum. More preferably, the grain comminution further comprises malted barley.

[0015] Optionally, an additional amount of a rye-inhibited xylanase is added during any of steps (b), (c), and / or (d). Optionally, the method further comprises the additional step of adding an alpha-amylase and / or a beta-glucanase in any of steps (b), (c), (d), and / or (e). Optionally, a beta-glucanase is added. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 8.

[0016] In the process of the present disclosure, optionally a glucoamylase is added in step (f) and / or a protease is added in step (g).

[0017] Optionally, the method further comprises the step of adding an alpha-L-arabinofuranosidase in any of steps (b), (c), (d) and / or (e). Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence with at least 70% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence with at least 80% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence with at least 85% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence with at least 90% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence with at least 92% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence with at least 95% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence with at least 98% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity, having a polypeptide amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.

[0018] The method of the present disclosure also optionally comprises the additional step of adding feruloyl esterase in any of steps (b), (c), (d), and / or (e). Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 70% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 92% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

[0019] In another aspect of the application, a method for preparing a low viscosity mash is presented, the method having the steps of: (a) preparing a mash from a cereal grind having rye grain in the presence of a xylanase having reduced sensitivity to rye XIP inhibitors; (b) optionally filtering the mash to obtain wort. Optionally, the xylanase is inhibited by rye XIP inhibitors by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1%. Optionally, the xylanase is inhibited by rye XIP inhibitors by less than 20%.

[0020] Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 3.

[0021] Optionally, the rye kernels are malted. Optionally, the rye kernels are unmalted. Optionally, the rye kernels are milled. Optionally, the cereal grind is 60, 70, 80, 85, 90, 95, 99, or 100 w / w% rye. In other preferred embodiments, the cereal grind further comprises malted or unmalted barley, wheat, corn, rye, rice, cassava, oat flakes, or sorghum. Optionally, the cereal grind further comprises malted barley.

[0022] Optionally, the method further comprises the additional step of adding an alpha-amylase and / or a beta-glucanase to the wort. Optionally, a beta-glucanase is added. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 8.

[0023] Optionally, the method further comprises the step of adding an alpha-L-arabinofuranosidase to the mash. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence that has at least 70% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence that has at least 92% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity, having a polypeptide amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.

[0024] The method of the present disclosure also optionally includes the additional step of adding a feruloyl esterase to the wort. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 70% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 92% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 98% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

[0025] In another aspect of the application, a method for identifying a xylanase having reduced sensitivity to a cereal XIP inhibitor is presented, the method having the steps of determining the activity of the xylanase in the presence and absence of the cereal XIP inhibitor, and determining the percentage inhibition of the cereal XIP inhibitor. Optionally, the cereal is barley, wheat, maize, rye, rice, cassava, oat flakes or sorghum. Optionally, the cereal is rye or wheat. Optionally, the cereal is rye. Optionally, the cereal is wheat.

[0026] In another aspect of the application, a xylanase having reduced sensitivity to a rye XIP inhibitor, identified by the above method, is presented. Optionally, the xylanase is inhibited by the rye XIP inhibitor less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1%. Optionally, the xylanase is inhibited by the rye XIP inhibitor less than 20%.

[0027] Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 3.

[0028] In another aspect of the application, a xylanase is presented having reduced sensitivity to wheat XIP inhibitor identified by the method of claim 110. Optionally, the xylanase is inhibited by less than 90%, 80%, 70%, 60%, 55%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% by the wheat XIP inhibitor. Optionally, the xylanase is inhibited by less than 55% by the wheat XIP inhibitor.

[0029] Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 3.

[0030] In another aspect of the application, a composition for reducing wort viscosity in wheat adjunct brewing is presented, the composition having a beta-glucanase and a xylanase having reduced sensitivity to wheat XIP inhibitors. Optionally, the xylanase is an enzyme having xylanase activity, having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity, having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity, having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity, having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity, having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof, and wherein the beta- glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof, and wherein the beta- glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof, and wherein the beta- glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, and wherein the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 8.

[0031] In another aspect of the application, a process for preparing a low viscosity mash is presented, the process having the steps of: (a) preparing a mash from a cereal comminution having wheat kernels in the presence of a composition having a beta-glucanase as described above and a xylanase having reduced sensitivity to wheat XIP inhibitors; (b) optionally filtering the mash to obtain wort.

[0032] Optionally, the wheat kernels are malted. Optionally, the wheat kernels are unmalted. Optionally, the wheat kernels are ground. Optionally, the grain comminution is 5, 10, 20, 25, 30, 35, 40, 45, 50, 51, 60, 70, 80, 85, 90, 95, 99, or 100 w / w% wheat. Optionally, the grain comminution further has malted or unmalted barley, corn, rye, rice, cassava, oat flakes, or sorghum. Optionally, the grain comminution further has malted barley.

[0033] BIOLOGICAL SEQUENCE SUMMARY

[0034] SEQ ID NO: 1 shows the protein sequence of the FveXyn4_l precursor protein.

[0035] SEQ ID NO: 2 shows the protein sequence of the FveXyn4_l mature protein.

[0036] SEQ ID NO: 3 shows the protein sequence of the FveXyn4_l predicted protein.

[0037] SEQ ID NO: 4 shows the amino acid sequence of the PfunXyn GH11 xylanase (E.C. 3.2.1.8) precursor protein.

[0038] SEQ ID NO: 5 shows the amino acid sequence of the PfunXyn GH11 xylanase (E.C. 3.2.1.8) mature protein.

[0039] SEQ ID NO: 6 shows the amino acid sequence of the TemXyn GH10 xylanase (E.C. 3.2.1.8) precursor protein.

[0040] SEQ ID NO: 7 shows the amino acid sequence of the TemXyn GH10 xylanase (E.C. 3.2.1.8) mature protein.

[0041] SEQ ID NO: 8 shows the amino acid sequence of the b-glucanase BsbBglu mature protein (E.C. 3.2.1.6) from Bacillus subtilis.

[0042] SEQ ID NO: 9 shows the amino acid sequence of the xylanase variant AkaXyl mature protein (E.C. 3.2.1.8) from Aspergillus kawachii.

[0043] SEQ ID NO: 10 shows the amino acid sequence of the AniAfur precursor protein alpha-L-arabinofuranosidase.

[0044] SEQ ID NO: 11 shows the amino acid sequence of the BaAfur precursor protein a-L-arabinofuranosidase.

[0045] SEQ ID NO: 12 shows the amino acid sequence of the CtFe precursor protein ferulic acid esterase.

[0046] SEQ ID NO: 13 shows the DNA sequence of primer 1.

[0047] SEQ ID NO: 14 shows the DNA sequence of primer 2. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 Turbidity (turbidity) quantified as haze in EBC units (small particles) by Anton Paar by 90° scattering of wort supernatant is depicted. Haze scattering of experiments 3 and 6, 4 and 7, and 5 and 8, respectively, with PfunXyn or FvXyn4_1 xylanases (in combination with a-L-arabinofuranosidase and ferulic acid esterase) as indicated in the legend is shown.

[0049] Figure 2 Turbidity quantified as haze in EBC units (larger particles) by Anton Paar by 25° scattering of wort supernatant is depicted. Haze scattering of experiments 3 and 6, 4 and 7, and 5 and 8, respectively, with PfunXyn or FvXyn4_1 xylanases (in combination with a-L-arabinofuranosidase and ferulic acid esterase) as indicated in the legend is shown.

[0050] Figure 3 Average wort volume collected after 30 minutes of filtration at the following dose ranges for various xylanases is depicted: TemXyn applied at 0.27 to 0.69 U / g cereal mash and FveXyn4_1 applied at 9.0 to 27.2 pg FveXyn4_1 protein / g cereal mash.

[0051] Figure 4 Average wort viscosity (mPa.s) at 12°P after filtration using the following doses for various xylanases is depicted: TemXyn applied at 0.27 to 0.69 U / g cereal mash and FveXyn4_1 applied at 9.0 to 27.2 pg FveXyn4_1 protein / g cereal mash.

[0052] Figure 5The average initial extract concentration (Original Extract) (°P) of the filtered wort is depicted using various xylanases at the following doses: TemXyn was applied at 0.27 to 0.69 U / g of cereal ground material and FveXyn4_1 was applied at 9.0 to 27.2 pg FveXyn4_1 protein / g of cereal ground material.

[0053] Figure 6 The effect of increasing xylanase (Viscoferm, PfunXyn and FveXyn4_1) application rate (g / kg of cereal ground material) on ethanol production in a rye-based SSF fermentation is depicted. DETAILED DESCRIPTION

[0054] DEFINITIONS

[0055] An "active fragment" of an enzyme is a polypeptide in which the enzyme has had deletions at the C-terminus, N-terminus, and / or internally, but which still retains some or all of its original activity. Active fragments include the mature form of the enzyme.

[0056] In addition to the specific amino acid sequences and polynucleotides mentioned herein, the present application encompasses variants, homologues, derivatives, and fragments thereof.

[0057] The term "variant" is used to mean a nucleotide sequence or an amino acid sequence that is different from the wild-type sequence.

[0058] For example, a variant can include a substitution, an insertion, a deletion, a truncation, a transversion, and / or an inversion at one or more positions relative to the wild-type sequence. Variants can be prepared using methods known in the art, such as site-scan mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed evolution, as well as using recombination methods well known in the art. Polynucleotide sequences encoding variant amino acid sequences can be readily synthesized using methods known in the art.

[0059] In some aspects, a variant is a naturally-occurring nucleotide sequence or amino acid sequence that is different from the wild-type sequence. For example, a variant can be a naturally-occurring genetic variant.

[0060] In some aspects, a variant is an engineered variant. For example, a variant can be engineered by recombinant methods.

[0061] The subject protein sequences of the present application can also have deletions, insertions, or substitutions of amino acid residues which generate silent changes and result in a functionally equivalent substance. Amino acid substitutions can be deliberate, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids which are hydrophilic, neutral, include cysteine, serine, threonine, glycine, alanine, asparagine, glutamine, phenylalanine, and tyrosine.

[0062] Conservative substitutions can be made, for example, according to the following table. Amino acids in the same block in Column 2, and preferably amino acids in the same block in Column 3, can be substituted for one another as shown in Table 1.

[0063] Table 1

[0064]

[0065] The present application also encompasses homologous substitutions (substitutions and replacements are used herein to mean the exchange of an existing amino acid residue for an alternative residue), i.e. like-for-like substitutions, such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitutions can also occur, i.e. from one class of residue to another or alternatively involving non-natural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyridylalanine, thiophenylalanine, naphthylalanine and phenylglycine.

[0066] Substitutions can also be made by synthetic amino acids (e.g. non-natural amino acids) including: alpha and alpha-disubstitutions amino acids, N-alkyl amino acids , lactic acid , halide derivatives of natural amino acids (such as trifluorotyrosine , p-Cl-phenylalanine , p-Br-phenylalanine , p-I-phenylalanine , L-allyl-glycine , ß-alanine , L-a-aminobutyric acid , L-g-aminobutyric acid , L-a-aminoisobutyric acid , L-e-aminohexanoic acid # , 7-aminoheptanoic acid , L-methionine sulfone # L-norleucine L-norvaline p-nitro-L-phenylalanine L-hydroxyproline # L-thioproline methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe penta-methyl-Phe L-Phe (4-amino) # L-Tyr (methyl) L-Phe (4-isopropyl) L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) L-diaminopropionic acid # and L-Phe (4-benzyl) .

[0067] For the purposes of the above discussion (in relation to homologous or non-homologous substitution), the symbol is used to refer to the hydrophobic nature of the derivative, whereas # is used to refer to the hydrophilic nature of the derivative, refers to the amphipathic character.

[0068] Variant amino acid sequences can comprise suitable spacer groups which can be inserted between any two amino acid residues of the sequence, including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or b-alanine residues. Further variations (involving the presence of one or more amino acid residues in peptoid form) will be well understood by persons skilled in the art. For the avoidance of doubt, "peptoid form" is used to refer to variant amino acid residues in which the a-carbon substituent group is on the nitrogen atom of the residue rather than on the a-carbon. Methods for preparing peptides in peptoid form are known in the art, for example Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

[0069] Other variants of the sequences described herein can be obtained, for example, by probing DNA libraries prepared from a range of individuals (e.g., individuals from different populations). In addition, other homologues can be obtained and such homologues, and fragments thereof, will generally be capable of selective hybridization to the sequences shown in the sequence listing herein. Such sequences can be obtained by probing cDNA libraries or genomic DNA libraries prepared from other animal species and probing such libraries with probes comprising all or a portion of any of the sequences contained in the attached sequence listing under conditions of moderate to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the application.

[0070] Variants and strain / species homologues can also be obtained using degenerate PCR, which will use primers designed to target sequences within the variants and homologues that encode conserved amino acid sequences within the sequences of the application. The conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants / homologues. Sequence alignments can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used.

[0071] The primers used in the degenerate PCR will contain one or more degenerate positions and will be used under conditions of lower stringency than those used to clone sequences from known sequences with single sequence primers.

[0072] Alternatively, such polynucleotides can be obtained by site-directed mutagenesis of a characterized sequence. This can be useful, for example, where silent codon sequence changes are needed to optimize the codon bias of a particular host cell in which the polynucleotide sequence is expressed. Other sequence changes can be desirable in order to introduce restriction endonuclease recognition sites, or in order to alter the properties or function of the polypeptide encoded by the polynucleotide.

[0073] Unless otherwise indicated, the present application employs conventional biochemistry, molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, 1-3 volumes, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, Chapters 9, 13, and 16, John Wiley & Sons, New York, NY); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press, each of which is incorporated herein by reference.

[0074] As used herein, "percent (%) sequence identity" means that a specified sequence has at least a certain percentage of amino acid residues identical with the amino acid residues in a specified reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. The default parameters for the CLUSTAL W algorithm are:

[0075] Gap open penalty: 10.0

[0076] Gap extension penalty: 0.05

[0077] Protein weight matrix: BLOSUM series

[0078] DNA weight matrix: IUB

[0079] Delay divergent sequences %: 40

[0080] Gap separation distance: 8

[0081] DNA transition weight: 0.50

[0082] List of hydrophilic residues: GPSNDQEKR

[0083] Use negative matrix: off

[0084] Switch special residue penalties: on

[0085] Switch hydrophilic penalties: on

[0086] Switch end gap separation penalties: off

[0087] Deletions are considered non-identical residues as compared to the reference sequence. Deletions occurring at either end are included. For example, a variant having a five amino acid deletion at the C-terminus of a mature 617 residue polypeptide would have 99% percent sequence identity (612 / 617 identical residues x 100, rounded to the nearest integer) relative to the mature polypeptide. Such a variant would be encompassed by a variant having "at least 99% sequence identity" to the mature polypeptide.

[0088] According to the present application, the proteins (including enzymes) of the present application exist in a variety of forms. The proteins of the present application can be truncated or clipped (i.e., amino acids removed) from the N-terminus and / or C-terminus, resulting in shorter proteins. The proteins of the present application can also have internal deletions. Shorter proteins as described herein can have higher activity or lower activity than their longer counterparts. Without being bound by theory, as used herein, the term precursor protein is a protein (including enzyme) that has an N-terminal signal peptide that targets the protein for secretion. Precursor proteins are sometimes referred to herein as "full-length" or "full-length proteins." The N-terminal signal peptide is cleaved in the endoplasmic reticulum to produce the mature protein.

[0089] Xylanases are classified as EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.136, and EC 3.2.1.156; activity thereof can be measured, for example, as described in the Examples. Suitable xylanases to be used in combination with the enzymes according to the present application that exhibit endo-1,3(4)-beta-glucanase activity include any xylanase classified as EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.136, and EC 3.2.1.156, for example, any xylanase disclosed in WO 2010072226, WO 2010072225, WO 2010072224, WO 2005059084, WO 2007056321, WO 2008023060 A, WO 9421785, WO 2006114095, WO 2006066582, US 2008233175, and WO 10059424.

[0090] Endo-beta-1,4-xylanase (also referred to herein as xylanase) is the name given to a class of enzymes that degrade the linear polysaccharide beta-1,4-xylan to xylose, thus breaking down hemicellulose, one of the major components of plant cell walls. Endo-1,4-beta xylanase is classified as EC 3.2.1.8. This enzyme causes endohydrolysis of 1,4-beta-D-xylosidic linkages in xylan.

[0091] The xylanases of the present application are superior for both the solubilization of insoluble arabinoxylan (WU-AX) and water-soluble arabinoxylan (WE-AX) of rye fiber. In addition, the xylanases of the present application have superior product properties and high thermal stability relevant to, for example, starch saccharification applications.

[0092] The term "family 10 xylanase", "glycoside hydrolase (GH) family 10" or simply "GH 10 xylanase" comprises enzymes with a number of known activities, such as xylanase (EC: 3.2.1.8); endo-1,3-beta-xylanase (EC: 3.2.1.32); cellobiohydrolase (EC: 3.2.1.91). These enzymes were formerly known as cellulase family F.

[0093] As used herein, the term "family 11 xylanase", "glycoside hydrolase (GH) family 11 " or simply "GH 11 xylanase" refers to endo-1,4-beta xylanases classified as EC 3.2.1.8, which bring about endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans, and which are classified as family 11 xylanases according to B. Henrissat, A classification of glycosyl hydrolases based on amino acid sequence similarities, Biochem. J. 280 (1991), pp. 309-316.

[0094] The xylanase variants described in the present invention are superior for the solubilization of both insoluble arabinoxylans (WU-AX) and water soluble arabinoxylans (WE-AX) of rye fibers. This property of the xylanase, when used during mashing of rye based material, provides enhanced filterability and / or improved extract yield after filtration.

[0095] All references cited in this specification are hereby incorporated by reference in their entirety. In particular, the disclosure of all references referred to herein are incorporated by reference.

[0096] Unless otherwise defined, all terms (including technical and scientific terms) used in the disclosure of the present invention have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0097] Definitions for other certain words and phrases are provided throughout this patent document. To the extent a term or phrase is not expressly defined, it should be given its broadest context interpretation.

[0098] Additional definitions are set forth below.

[0099] Production of enzymes

[0100] The enzymes of the present application can be produced in host cells, for example, by secretion or intracellular expression. Following secretion of the enzyme into the cell culture medium, a culture cell material having the enzyme (e.g., whole cell broth) can be obtained. Optionally, the enzyme can be isolated from the host cell, or even from the cell culture broth, depending on the desired purity of the final enzyme. Suitable host cells include bacteria, fungi (including yeast and filamentous fungi), and plant cells (including algae). Particularly useful host cells include Aspergillus niger, Aspergillus oryzae, or Trichoderma reesei. Other host cells include bacterial cells, such as B. subtilis or B. licheniformis, as well as Streptomyces, E. coli.

[0101] Vectors

[0102] DNA constructs comprising nucleic acids encoding enzymes can be constructed for expression in host cells. Due to the well-known degeneracy of the genetic code, variant polynucleotides encoding the same amino acid sequence can be designed and prepared using routine techniques. Codon optimization for a particular host cell is also well known in the art. Nucleic acids encoding xylanases can be incorporated into vectors. The vectors can be transferred into host cells using well-known transformation techniques, such as those disclosed below.

[0103] Vectors The vectors can be any vector that can be transformed into a host cell and replicated within the host cell. For example, vectors comprising nucleic acids encoding enzymes can be transformed into bacterial host cells and replicated therein as a means of propagating and amplifying the vectors. The vectors can also be transformed into expression hosts such that the encoding nucleic acids can be expressed as functional xylanases. Host cells used as expression hosts can include, for example, filamentous fungi. The strain catalog of the American Fungal Genetics Stock Center (FGSC) lists vectors suitable for use in expressing in fungal host cells. See FGSC, Fungal Genetics Stock Center [Strain Catalog], University of Missouri, in www.fgsc.net (last modified January 17, 2007). A representative vector is pJG153, which is a promoterless Cre expression vector that can be replicated in bacterial hosts. See Harrison et al., (June 2011) Applied Environ. Microbiol 77: 3916-22. pJG153 can be modified using routine techniques to comprise and express nucleic acids encoding xylanases.

[0104] The nucleic acid encoding the enzyme can be operably linked to a suitable promoter that allows transcription in the host cell. The promoter can be any DNA sequence that shows transcriptional activity in the host cell of choice and can be derived from genes either homologous to the host cell or foreign to the host cell. Examples of suitable promoters for directing the transcription of the DNA sequences encoding the xylanase, especially in a bacterial host, are the promoter of the lactose operon of E. coli, the Streptomyces coelicolor agarase gene dagA or celA promoter, the promoters of the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes, and the like. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, or Aspergillus nidulans acetamidase. When the gene encoding the enzyme is expressed in a bacterial species, such as E. coli, a suitable promoter can be selected, for example, from bacteriophage promoters including T7, and phage lambda promoters, when the gene encoding the enzyme is expressed in a yeast species, examples of suitable promoters include, but are not limited to, the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the AOX1 or AOX2 promoters of Pichia pastoris. cbh1 is an endogenous inducible promoter from T. reesei. See Liu et al., (2008) “Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbh1) promoter optimization,” Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.

[0105] The coding sequences can be operably linked to a signal sequence. The DNA encoding the signal sequence can be a DNA sequence naturally associated with the enzyme gene to be expressed or from a different genus or species. The signal sequence and the promoter sequence making up the DNA construct or vector can be introduced into the fungal host cell and can be derived from the same source. For example, the signal sequence is the cbhl signal sequence operably linked to the cbhl promoter.

[0106] The expression vector can also contain a suitable transcription terminator, and in eukaryotes, a polyadenylation sequence operably linked to the DNA sequence encoding the variant xylanase. The terminator and polyadenylation sequences can be appropriately derived from the same sources as the promoter.

[0107] The vector can further contain a DNA sequence enabling autonomous replication of the vector in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMB1, and pIJ702.

[0108] The vector can also contain a selectable marker, e.g., a gene whose product complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance (e.g., ampicillin, kanamycin, chloramphenicol, or tetracycline resistance, for example). In addition, the vector can contain an Aspergillus selection marker (such as amdS, argB, niaD, and xxsC), a marker that confers hygromycin resistance, or selection can be achieved by co- transformation, as is known in the art. See, e.g., International PCT Application WO 91 / 17243.

[0109] Intracellular expression can be advantageous in some aspects, e.g., when using certain bacteria or fungi as host cells to produce large quantities of xylanase for subsequent enrichment or purification. Extracellular secretion of xylanase into the culture medium can also be used to prepare culture cell material comprising isolated xylanase.

[0110] Expression vectors typically include components of cloning vectors, such as, for example, elements that allow the vector to replicate autonomously in a selected host organism and one or more phenotypically detectable markers for selection purposes. Expression vectors usually comprise control nucleotide sequences, such as promoters, operators, ribosome binding sites, translation initiation signals, and optionally repressor or one or more activator genes. In addition, the expression vector can comprise a sequence encoding an amino acid sequence that is capable of targeting the enzyme to a host cell organelle (such as peroxisomes) or to a specific host cell compartment. Such targeting sequences include, but are not limited to, the sequence SKL. For expression under the direction of a control sequence, the nucleic acid sequence of the xylanase is operably linked to the control sequence in a suitable manner with respect to expression.

[0111] The procedures used to ligate the DNA construct encoding the enzyme, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication are well known to persons skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory, 1989, and 3rded., 2001).

[0112] Transformation and cultivation of host cells

[0113] Isolated cells comprising the DNA construct or expression vector are advantageously used as host cells for the recombinant production of the enzyme according to the application. The cells can be transformed with the DNA construct encoding the enzyme (in one or more copies) conveniently by integrating the DNA construct into the host chromosome. It is generally considered advantageous if the integration is effected, because the DNA sequence is then more likely to be stably maintained in the cell. The integration of the DNA construct into the host chromosome can be carried out in accordance with conventional methods, for example by homologous or heterologous recombination. Alternatively, the cells can be transformed with expression vectors associated with different types of host cells as described above.

[0114] Examples of suitable bacterial host organisms are Gram-positive bacterial species, such as Bacillaceae, including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus stearothermophilus (formerly Bacillus stearothermophilus), Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis; Streptomyces species, such as Streptomyces murinus; lactic acid bacterial species, including Lactococcus sp., such as Lactococcus lactis; Lactobacillus sp., including Lactobacillus reuteri; Leuconostoc sp.; Pediococcus sp.; and Streptococcus sp. Alternatively, strains of Gram-negative bacterial species belonging to the family Enterobacteriaceae (including Escherichia coli) or to the family Pseudomonadaceae can be chosen as host organisms.

[0115] Suitable yeast host organisms can be selected from biotechnology relevant yeast species such as, but not limited to, yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces, or Saccharomyces species (including S. cerevisiae), or species belonging to the genus Schizosaccharomyces (such as, for example, S. pombe). The methylotrophic yeast species strain Pichia pastoris can be used as a host organism. Alternatively, the host organism can be a Hansenula sp. Suitable host organisms among filamentous fungi include Aspergillus sp., such as, for example, Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively, strains of Fusarium sp. (for example, Fusarium oxysporum) or Rhizomucor sp. (such as Rhizomucor miehei) can be used as host organisms. Other suitable strains include Thermomyces and Mucor sp. Furthermore, Trichoderma sp. can be used as a host. Suitable procedures for transforming Aspergillus host cells include, for example, the procedures described in EP 238023. Enzymes expressed by the fungal host cells can be glycosylated, i.e., comprise a glycosyl moiety. The glycosylation pattern can be the same as or different from that present in wild-type xylanase. The type and / or extent of glycosylation can confer changes in enzymatic and / or biochemical properties.

[0116] It can be advantageous to delete genes from the expression host, where the genetic deficiency can be cured by the transforming expression vector. Known methods can be used to obtain fungal host cells having one or more inactivated genes. Gene inactivation can be accomplished by complete or partial deletion, by insertion inactivation, or by any other means that prevents the gene from expressing a functional protein. Any of the cloned genes from Trichoderma sp. or other filamentous fungal hosts can be deleted, for example, the cbhl, cbh2, egll, and egl2 genes. Gene deletion can be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.

[0117] Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, for example, lipofection-mediated and DEAE-dextrin-mediated transfection; incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art. See, e.g., Sambrook et al. (2001), supra. Expression of heterologous proteins in Trichoderma is described, e.g., in U.S. Patent No. 6,022,725. For transformation of Aspergillus strains, reference is also made to Cao et al. (2000) Science 9:991-1001. The vector system can be used to construct a genetically stable transformant, whereby the nucleic acid encoding the enzyme is stably integrated into the host cell chromosome. The transformant is then selected and purified by known techniques.

[0118] Expression

[0119] Methods of producing the enzymes of the present application can include culturing a host cell as described above under conditions conducive to production of the enzyme, and recovering the enzyme from the cells and / or culture medium.

[0120] The culture medium used to cultivate the cells can be any conventional medium suitable for growing the host cell under consideration and obtaining expression of the enzyme. Suitable culture media and medium components are available from commercial suppliers or can be prepared according to published recipes (e.g., as described in the catalog of the American Type Culture Collection).

[0121] The enzyme secreted from the host cell can be used in a whole broth preparation. In the methods of the present application, preparation of a used whole fermentation broth of a recombinant microorganism resulting in expression of a xylanase enzyme can be achieved using any cultivation method known in the art. Thus, fermentation is understood to include shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions promoting expression or secretion of the xylanase enzyme. The term "used whole fermentation broth" is defined herein to include unfractionated contents of the fermentation material, including culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is understood that the term "used whole fermentation broth" also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.

[0122] The enzyme secreted from the host cell can be recovered from the culture medium by means of well-known procedures, including separating the cells from the culture medium by centrifugation or filtration, and precipitating the proteinaceous components of the culture medium with a salt, such as ammonium sulfate, followed by use of chromatographic procedures such as ion exchange, affinity, etc.

[0123] The host cells can be cultured under suitable conditions that allow for expression of the xylanases. Expression of these enzymes can be constitutive, such that they are produced continuously, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when desired by, for example, adding an inducing substance (e.g., dexamethasone or IPTG or sophorose) to the culture medium. Polypeptides can also be produced recombinantly in in vitro cell-free systems (such as the TNT™ (Promega) rabbit reticulocyte system).

[0124] Methods for enriching and purifying enzymes

[0125] Fermentation, isolation and concentration techniques are well known in the art and can be used to prepare a solution containing the enzyme polypeptide using conventional methods.

[0126] Following fermentation, a fermentation broth is obtained and microbial cells and various suspended solids, including residual raw fermentation material, are removed by conventional separation techniques in order to obtain an enzyme solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, post-centrifugation ultrafiltration, extraction or chromatography, among others, are commonly used.

[0127] It is desirable to concentrate the solution containing the enzyme polypeptide to optimize recovery. The use of unconcentrated solutions requires increased incubation times in order to collect the enriched or purified enzyme precipitate.

[0128] The enzyme-containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme-containing solution can be achieved by any of the techniques discussed herein. Exemplary methods of enrichment and purification include, but are not limited to, rotary vacuum filtration and / or ultrafiltration.

[0129] Description of preferred embodiments

[0130] According to one aspect of the present invention, it has been discovered that xylanases having reduced sensitivity to xylanase inhibitors in adjuncts, such as rye and wheat, can be used in distillation and brewing to overcome long-standing problems. U.S. law requires that spirits sold as rye whiskey must be produced from malt made up of at least 51% rye kernels. Those skilled in the art know that such malts are very difficult to work with on an industrial scale.

[0131] The present invention relates to a rye starch saccharification and optional filtration step in a process for producing an alcoholic beverage, such as beer, or for distillation applications, such as whiskey, and to a composition useful in a starch saccharification and optional filtration step in such a process.

[0132] The use of xylanases to improve the filterability of malt-based worts is known. However, according to the present invention, it has been found that specific protein inhibitors of xylanases are present in non-malt adjuncts such as rye and wheat. According to the present invention there is provided a method for screening xylanases for those that are less inhibited by the wheat and rye factors.

[0133] Disclosed are xylanases that have very low inhibition by rye and wheat XIP, and show superior properties in reducing the viscosity of rye and wheat based worts. In beer brewing, the wort is usually filtered to produce a liquid wort for subsequent operations. Here, the reduction in viscosity improves filterability. In distilling and in some beer brewing, the wort is fermented without filtration. Here, the reduction in wort viscosity reduces the degree of fouling of the fermentation and distilling equipment and increases the ethanol yield.

[0134] According to an aspect of the present invention, there is provided a method for producing a rye whiskey, the method having the steps of: (a) providing a cereal mash comprising 51 w / w% of rye kernels; (b) adding water to the cereal mash to provide a wort; (c) subjecting the wort of step (b) to pre-liquefaction; (d) gelatinizing the wort of step (c); (e) subjecting the wort of step (d) to liquefaction in the presence of a xylanase having reduced sensitivity to rye XIP inhibitors; (f) subjecting the wort of step (e) to saccharification; (g) fermenting the wort of step (f) to produce a fermentate containing ethanol; (h) distilling the fermentate of step (g) to provide a rye whiskey. Preferably, the xylanase is inhibited by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% by the rye XIP inhibitor. More preferably, the xylanase is inhibited by less than 20% by the rye XIP inhibitor.

[0135] Preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Still more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Yet more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. In still more preferred embodiments, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. In still more preferred embodiments, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Yet more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. In still more preferred embodiments, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Most preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 3.

[0136] Preferably, the rye kernels are malted. In other preferred embodiments, the rye kernels are unmalted. Preferably, the rye kernels are milled. Preferably, the cereal grind is 60, 70, 80, 85, 90, 95, 99, or 100 w / w% rye. In other preferred embodiments, the cereal grind further comprises malted or unmalted barley, wheat, corn, rye, rice, cassava, oat flakes, or sorghum. More preferably, the cereal grind further comprises malted barley.

[0137] Preferably, an additional amount of a non-rye-inhibited xylanase is added during any of steps (b), (c) and / or (d). Preferably, the method further comprises the additional step of adding an alpha-amylase and / or a beta-glucanase in any of steps (b), (c), (d) and / or (e). More preferably, a beta-glucanase is added. Preferably, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. More preferably, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Still more preferably, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Yet more preferably, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. In still more preferred embodiments, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. More preferably, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Still more preferably, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Yet more preferably, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. In still more preferred embodiments, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Most preferably, the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 8.

[0138] In the methods of the present disclosure, a glucoamylase is preferably added in step (f) and / or a protease is added in step (g).

[0139] Preferably, the method further comprises the step of adding an alpha-L-arabinofuranosidase in any of steps (b), (c), (d) and / or (e). Preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 70% sequence identity with SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. More preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 80% sequence identity with SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Still more preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 85% sequence identity with SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Yet more preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. In still more preferred embodiments, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 92% sequence identity with SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. More preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 95% sequence identity with SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Yet more preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 98% sequence identity with SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.Still more preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. In the most preferred embodiment, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.

[0140] The method of the present disclosure also preferably comprises the additional step of adding a feruloyl esterase in any of steps (b), (c), (d) and / or (e). Preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. More preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Still more preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Yet more preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Still more preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Yet more preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. In still more preferred embodiments, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. More preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Most preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

[0141] In another aspect of the application, a method for preparing a low viscosity mash is presented, the method having the steps of: (a) preparing a mash from a cereal grind having rye grain in the presence of a xylanase having reduced sensitivity to rye XIP inhibitors; (b) optionally filtering the mash to obtain wort. Preferably, the xylanase is inhibited by rye XIP inhibitors by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1%. More preferably, the xylanase is inhibited by rye XIP inhibitors by less than 20%.

[0142] Preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Still more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Yet more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. In still more preferred embodiments, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. In still more preferred embodiments, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Yet more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. In still more preferred embodiments, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Most preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 3.

[0143] Preferably, the rye kernels are malted. In other preferred embodiments, the rye kernels are unmalted. Preferably, the rye kernels are milled. Preferably, the cereal grind is 60, 70, 80, 85, 90, 95, 99, or 100 w / w% rye. In other preferred embodiments, the cereal grind further comprises malted or unmalted barley, wheat, corn, rye, rice, cassava, oat flakes, or sorghum. More preferably, the cereal grind further comprises malted barley.

[0144] Preferably, the method further comprises the additional step of adding an alpha-amylase and / or a beta-glucanase to the mash. More preferably, a beta-glucanase is added. Preferably, the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. More preferably, the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Still more preferably, the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Yet more preferably, the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. In still more preferred embodiments, the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. More preferably, the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Still more preferably, the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Yet more preferably, the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. In still more preferred embodiments, the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Most preferably, the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 8.

[0145] Preferably, the method further comprises the step of adding an alpha-L-arabinofuranosidase to the mash. Preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. More preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Still more preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Yet more preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. In still more preferred embodiments, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. More preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. Yet more preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.Still more preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity, having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof. In the most preferred embodiment, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.

[0146] The method of the present disclosure also preferably comprises the further step of adding a feruloyl esterase to the wort. Preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. More preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Still more preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Yet more preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Still more preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Yet more preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. In still more preferred embodiments, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. More preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Most preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity, having a polypeptide amino acid sequence according to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

[0147] In another aspect of the application, a method for identifying a xylanase having reduced sensitivity to a cereal XIP inhibitor is presented, the method having: determining the activity of the xylanase in the presence and absence of the cereal XIP inhibitor, and determining the percentage inhibition of the cereal XIP inhibitor. Preferably, the cereal is barley, wheat, maize, rye, rice, cassava, oat flakes or sorghum. More preferably, the cereal is rye or wheat. Still more preferably, the cereal is rye. In other preferred embodiments, the cereal is wheat.

[0148] In another aspect of the application, a xylanase having reduced sensitivity to a rye XIP inhibitor identified by the above method is presented. Preferably, the xylanase is inhibited by the rye XIP inhibitor by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1%. Preferably, the xylanase is inhibited by the rye XIP inhibitor by less than 20%.

[0149] Preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Still more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Yet more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. In still more preferred embodiments, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. In still more preferred embodiments, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Yet more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. In still more preferred embodiments, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Most preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 3.

[0150] In another aspect of the application, a xylanase is presented having a reduced sensitivity to wheat XIP inhibitors identified by the method as claimed in claim 110. Preferably, the xylanase is inhibited by less than 90%, 80%, 70%, 60%, 55%, 50%, 40%, 30%, 20%, 10%, 5% or 1% by a wheat XIP inhibitor. More preferably, the xylanase is inhibited by less than 55% by a wheat XIP inhibitor.

[0151] Preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Still more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Yet more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. In still more preferred embodiments, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. In still more preferred embodiments, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Yet more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. In still more preferred embodiments, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof. Most preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 3.

[0152] In another aspect of the application, a composition for reducing wort viscosity in wheat adjunct brewing is presented, the composition having a beta-glucanase and a xylanase having reduced sensitivity to wheat XIP inhibitors. Preferably, the xylanase is an enzyme having xylanase activity, having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity, having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Still more preferably, the xylanase is an enzyme having xylanase activity, having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Yet more preferably, the xylanase is an enzyme having xylanase activity, having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. In still more preferred embodiments, the xylanase is an enzyme having xylanase activity, having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity, having a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.Yet more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof, and wherein the beta- glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof, and wherein the beta- glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof. Yet more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof, and wherein the beta- glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof. Still more preferably, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof. In the most preferred embodiment, the xylanase is an enzyme having xylanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, and wherein the beta-glucanase is an enzyme having beta-glucanase activity having a polypeptide amino acid sequence according to SEQ ID NO: 8.

[0153] In another aspect of the application, a process for preparing a low viscosity mash is presented, the process having the steps of: (a) preparing a mash from a cereal comminution having wheat kernels in the presence of a composition having a beta-glucanase as described above and a xylanase having reduced sensitivity to wheat XIP inhibitors; (b) optionally filtering the mash to obtain wort.

[0154] Preferably, the wheat kernels are malted. In other preferred embodiments, the wheat kernels are unmalted. Preferably, the wheat kernels are milled. Preferably, the cereal grind is 5, 10, 20, 25, 30, 35, 40, 45, 50, 51, 60, 70, 80, 85, 90, 95, 99, or 100 w / w% wheat. Preferably, the cereal grind also has malted or unmalted barley, corn, rye, rice, cassava, oat flakes, or sorghum. More preferably, the cereal grind also has malted barley.

[0155] Examples

[0156] The present disclosure is further described in the following examples, which are not intended to limit the scope of the disclosure claimed in any way. The accompanying drawings are intended to be part of the specification and description of the present disclosure. The following examples are provided to illustrate but not limit the claimed disclosure.

[0157] Example 1 - Enzymes

[0158] FveXyn4_1 is a mature xylanase variant from Fusarium verticilliodes having the amino acid sequence set forth in SEQ ID NO: 2.

[0159] As an example of a very effective xylanase for solubilization used in brewing, a preparation of GH11 xylanase (E.C. 3.2.1.8) from Penicillium funiculosum with the mature amino acid sequence shown in SEQ ID NO: 5 (PfunXyn) was used. The PfunXyn preparation (with an activity of 3150 CMC-DNS U / g) was provided by Dupont Nutrition Bioscience Aps (Denmark). As another example of a xylanase for solubilization effective for use in brewing, a preparation of GH10 xylanase (E.C. 3.2.1.8) from Talaromyces emersonii (Geosmithia emersonii) with the mature amino acid sequence shown in SEQ ID NO: 7 (TemXyn) was used. The TemXyn preparation (with an activity of 5525 units / g) was provided by Dupont Nutrition Bioscience Aps (Denmark). In addition, as an example of an enzyme complex that hydrolyzes beta-glucans and non-starch polysaccharides (like arabinoxylans), a preparation containing a b-glucanase (E.C. 3.2.1.6) from Bacillus subtilis with the mature amino acid sequence shown in SEQ ID NO: 8 (BsbBglu) and a xylanase variant (E.C. 3.2.1.8) from Aspergillus kawachi with the mature amino acid sequence shown in SEQ ID NO: 9 (AkaXyl_var) was used. The BsbBglu + AkaXyl_var preparation (with a xylanase activity of 13680 - 16720 XBU / g and with a b-glucanase activity of 9090 - 12500 BBU / g) was provided by Dupont Nutrition Bioscience Aps (Denmark). As an example of an enzyme complex with multiple cellulolytic activities, b-glucosidase (EC 3.2.1.21) and xylanase TrXyl produced by Trichoderma reesei (Hypocrea jecorina) were obtained from Dupont Nutrition Bioscience Aps (Denmark). b-glucanase from Bacillus subtilis (BsdBglu) (with a b-glucanase activity of 51000 U / g) was provided by Dupont Nutrition Bioscience Aps (Denmark).

[0160] As an example of an alpha-L-arabinofuranosidase with potential to hydrolyze terminal non-reducing alpha-L-arabinofuranose (alpha-1,2 > alpha-1,3) from monosubstituted xylose residues in arabinoxylan, alpha-L-arabinofuranosidase AniAfur from Aspergillus niger (89 U / mg 500 U) was sourced from Megazyme International Ireland Ltd. The optimal pH of AniAfur is 4.5, and the optimal temperature is 40 °C. As another example of an alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidase BaAfur from Bifidobacterium adolescentis (102 U / mg 400 U) was sourced from Megazyme International Ireland Ltd. The optimal pH of BaAfur is 6.0, and the optimal temperature is 50 °C. This enzyme is expected to hydrolyze alpha-1,3-linked L-arabinofuranose residues from the di-substituted D-xylosyl or L-arabinosyl residues of arabinoxylan and of pachyman, respectively, with high specificity.

[0161] As an example of a feruloyl esterase, feruloyl esterase CtFe from Clostridium thermocellum (0.5 U / mg 100 U) was sourced from Megazyme International Ireland Ltd. The optimal pH of CtFe is 6.0, and the optimal temperature is 60 °C. CtFe is expected to catalyze the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl) groups from esterified sugars (typically arabinose in "native" substrates).

[0162] Example 2 - Cloning of Fusarium verticillioides xylanase (FveXyn4)

[0163] Genomic DNA isolated from Fusarium verticillioides strain was used to amplify the xylanase gene. The sequence of the cloned gene, referred to as FveXyn4, was altered recombinantly as described in WO 2015 / 114108. The protein encoded by the FveXyn4_1 gene is described in SEQ ID No. 1. Based on PFAM search (http: / / pfam.sanger.ac.uk / ), the protein product of the gene FveXyn4 belongs to glycosyl hydrolase family 10 (GH10). At the N-terminal end, the FveXyn4 protein has a signal peptide of 25 amino acids predicted by SignalP-NN (Emanuelsson et al., Nature Protocols, 2:953-971, 2007). This indicates that FveXyn4 is a secreted glycosyl hydrolase.

[0164] Example 3 - Expression of Fusarium verticillioides xylanase variant FveXyn4_1

[0165] The FveXyn4_1 gene was amplified from genomic DNA of F. verticillioides variant 1 using primer 1 5'-caccATGAAGCTGTCTTCTTTCCTCTA-3' (SEQ ID NO: 13) and primer 2 5'-TTTTTAGCGGAGAGCGTTGACAACAGC-3' (SEQ ID NO: 14). The PCR product was cloned into the pENTR / D-TOPO vector (Invitrogen K2400) to generate the FveXyn4_1 pEntry plasmid. The expression plasmid pZZH254 was obtained by a Gateway Clonase II Enzyme Kit (Invitrogen 11791) Gateway Cloning reaction between the FveXyn4_1 pEntry plasmid and the pTrex3gM expression vector (described in US 2011 / 0136197 Al). The sequence of the FveXyn4 gene was confirmed by DNA sequencing. The plasmid pZZH254 was transformed into a quadruple deletion Trichoderma reesei strain (described in WO 05 / 001036) using biolistics (Te'o VS et al., J Microbiol Methods, 51 :393-9, 2002).

[0166] After sequence confirmation, protoplasts of the quadruple deletion Trichoderma reesei strain (described in WO 05 / 001036) were transformed with the expression plasmid pTTT-Ate CA1 using the PEG protoplast method (Penttila et al., Gene, 61 : 155-164, 1987). For protoplast preparation, spores were grown in Trichoderma minimal medium MM (20 g / L glucose, 15 g / L KH2PO4 (pH 4.5), 5 g / L (NH4)2SO4, 0.6 g / L MgSO4x7H2O, 0.6 g / L CaCl2x2H2O, 1 ml of 1000X Trichoderma reesei trace elements solution (175 g / L anhydrous citric acid, 200 g / L FeSO4x7H2O, 16 g / L ZnSO4x7H2O, 3.2 g / L CuSO4, 1.4 g / L MnSO4xH2O and 0.8 g / L boric acid)) at 24°C for about 10 hours. Germinated spores were harvested by centrifugation and treated with 30 mg / mL Vinoflow FCE (Novozymes AG, Switzerland) solution at 30°C for 7 hours to overnight at 100 rpm to lyse the fungal cell wall. Protoplasts were washed in 0.1 M Tris HCI buffer (pH 7) containing 0.6 M sorbitol and resuspended in 10 mM Tris HCI buffer (pH 7.5) containing 1.2 M sorbitol and 10 mM calcium chloride. For PEG transformation, approximately 1 pg of DNA and 1-5 x 107protoplasts (total volume 200 mΐ) were treated with 2 ml of 25% PEG solution, diluted with 2 volumes of 1.2 M sorbitol / 10 mM Tris (pH 7.5) / 10 mM CaCI2solution. Transformants were selected on medium containing acetamide as the sole nitrogen source (acetamide 0.6 g / L; cesium chloride 1.68 g / L; glucose 20 g / L; potassium phosphate monobasic 15 g / L; magnesium sulfate heptahydrate 0.6 g / L; calcium chloride dihydrate 0.6 g / L; iron (II) sulfate 5 mg / L; zinc sulfate 1.4 mg / L; cobalt (II) chloride 1 mg / L; manganese (II) sulfate 1.6 mg / L; agar 20 g / L; pH 4.25). Transformed colonies appeared in about 1 week (about 50-100). After growth on acetamide plates, spores were collected and reselected on acetamide plates. After 5 days, spores were collected using 10% glycerol and 1 x 108spores were inoculated in a 250 ml shake flask with 30 ml of glucose / sophorose limited medium for protein expression. Protein expression was confirmed by SDS-PAGE. Subsequently, spore suspensions were grown in a 7 L fermenter in limited medium containing 60% glucose-sophorose feed.Glucose / Glucofrangulin defined medium (per liter) consists of: (NH4)2SO4 5 g, PIPP'S buffer 33 g, Casamino acids 9 g, KH2PO4 4.5 g, CaCl2(anhydrous) 1 g, MgSO4.7H2O 1 g, pH adjusted to 5.5 with 50% NaOH, q.s. to 966.5 mL with Milli-Q H2O. After sterilization, the following are added: 26 mL of 60% glucose / glucofrangulin, and 2.5 mL of 400X T. reesei trace metals.

[0167] FveXyn4_1 was purified from concentrated fermentation broth of a 7 L fermenter culture using two chromatography columns. Concentrated fermentation broth buffered in 20 mM sodium phosphate buffer (pH 6.0) containing 1 M ammonium sulfate was loaded onto a hydrophobic interaction chromatography column (Sepharose Phenyl FF, 26 / 10). Protein was eluted from the column into 20 mM sodium phosphate buffer (pH 6.0) using a linear gradient of equilibration / wash buffer. Fractions containing FveXyn4_1 protein were loaded onto a gel filtration column (HiLoad Superdex 75 pg 26 / 60) and the mobile phase used was 20 mM sodium phosphate (pH 7.0) containing 0.15 M NaCl. Purified protein was concentrated using a 3 K Amicon Ultra-15 device and concentrated protein fractions were used for further studies.

[0168] Example 4 - Protein determination methods

[0169] Protein determination by standard Stain Free Imager Criterion

[0170] Proteins were quantified by SDS-PAGE gels and densitometry using a Gel Doc™ EZ imaging system. Reagents used in the assay: concentrated (2x) Laemmli sample buffer (Bio-Rad, Cat. No. 161-0737); 26-well XT 4%-12% Bis-Tris gels (Bio-Rad, Cat. No. 345-0125); Protein marker “Precision Plus Protein Standard” (Bio-Rad, Cat. No. 161-0363); Protein Standard BSA (Thermo Scientific, Cat. No. 23208) and SimplyBlue Safestain (Invitrogen, Cat. No. LC 6060). The assay was performed as follows: In a 96-well PCR plate, 50 µL of diluted enzyme sample was mixed with 50 µL of sample buffer containing 2.7 mg DTT. The plate was sealed with a Microseal ‘B’ film from Bio-Rad and placed in a PCR machine to heat to 70°C for 10 minutes. After that, the gel box was set by running the buffer fill chamber. Then 10 µL of each sample and standard (0.125-1.00 mg / mL BSA) was loaded onto the gel and 5 µL of marker was loaded. After that, the electrophoresis was run at 200 V for 45 min. After the electrophoresis, the gel was rinsed in water for 3 times for 5 min, then stained in Safestain overnight and finally destained in water. Then the gel was transferred to the imager. The intensity of each band was calculated using Image Lab software. A calibration curve was made using BSA (Thermo Scientific, Cat. No. 23208) and the amount of target protein was determined by band intensity and calibration curve. The protein quantification method was employed to prepare enzyme samples for subsequent examples.

[0171] Example 5 - Mature polypeptide sequence of F. verticillioides xylanase variant FveXyn4_1

[0172] The amino acid sequence of the predicted form of the FveXyn4_1 protein is shown in SEQ ID No. 3. This is expected to be the active form of the enzyme. However, we surprisingly identified that the mature and active form of the FveXyn4_1 protein is identical to the sequence shown in SEQ ID No. 2, likely due to post-translational modifications generated by the host during fermentation and endogenous activity affecting cleavage of the signal peptide.

[0173] Thus, it was surprisingly found that FveXyn4_1 was less proteolytically modified in the final fermentation broth at the end of fermentation compared to the predicted variant shown in SEQ ID NO: 3. The generated variants were verified using mass spectrometry as described in detail below. Thus, the N-terminus of the finally expressed variant was less processed compared to the 25 amino acid cleavage predicted by SignalP version 4.0, resulting in a final N-terminal truncation of 23 amino acids, which corresponds to the mature version of FveXyn4_1 having the polypeptide sequence of SEQ ID NO: 2. This mature variant of FveXyn4_1 has been used in all examples discussed herein.

[0174] To obtain the exact polypeptide sequence of the mature variant of FveXyn4_1, the protein band of the protein was cut from the SDS-PAGE gel and digested using three different enzymes (trypsin, A-chymotrypsin and Glu-C) to prepare the sample for mass spectrometry analysis. Trypsin specifically hydrolyzes the peptide bond on the carboxyl side of arginine I and lysine (K) residues, except when proline (P) is on the carboxyl side. A-chymotrypsin specifically hydrolyzes the peptide bond on the carboxyl side of tyrosine (Y), phenylalanine (F), tryptophan (W) and leucine (L), except when proline (P) is on the carboxyl side. Glu-C preferentially cleaves on the carboxyl side of glutamyl (E) in ammonium bicarbonate buffer at pH 8, but will also cleave on the carboxyl side of aspartyl (D) if hydrolysis is performed in phosphate buffer at pH 8.

[0175] To detect the exact C-terminus, the protein characterization (A2963) using the IFF program was used to prepare the protein of interest for analysis, where one change was the use of 40% 180-water in the digestion buffer. The proteolytic cleavage will thus incorporate 180- and 160-water into the resulting peptides, which will show as doublets in the MS spectrum. However, the protein C-terminus will only appear as a single peptide with 160-water, as it is not cleaved, but just the “last peptide” left by the protein. In this way, MS / MS analysis is used to map the C-terminus. To detect the exact protein N-terminus, the N-terminus of the intact protein was acetylation labeled prior to proteolytic digestion (IFF A manual 3448). The guanidination of lysine converts lysine to homoarginine and protects lysine (side chain) from acetylation. Only peptides originating from the N-terminus of the protein will be acetylated and thus unambiguously identified.

[0176] Example 6 - Viscosity reduction and filterability of different xylanases in combination with a-L-arabinofuranosidase and ferulic acid esterase in a rye-based starch saccharification trial

[0177] The purpose of this example is to demonstrate the benefit of non-inhibited xylanase during a starch saccharification process based on 100% rye. The enzymes were tested in a model system of a starch saccharification operation for wort production using milled rye (supplied by Hiram Walker Co., USA, material code: 202109-2201) (milled on a Buhler-Miag malt mill (0.5 mm setting, position 4)).

[0178] Starch saccharification procedure for wort production

[0179] Rye grain meal (60.0 g milled rye kernels) was mixed in a beaker with 203 g tap water in a starch saccharification bath (Lockner, LG-Electronics Division) cup and the pH was adjusted to pH 5.5 with 2.5 M sulfuric acid such that the ratio of water to grain meal was 3.38 : 1.

[0180] FveXyn4_1 xylanase variants were added based on the mg protein determined according to Example 4 in the following settings:

[0181] Test 1, 1.89 CMC-DNS U PfunXyn / g rye; Test 2, 6.8 pg FveXyn4_1 protein / g rye; Test 3, 1.89 CMC-DNS U PfunXyn / g rye + 0.3 U AniAfur / g rye + 0.3 U BaAfur / g rye; Test 4, 1.89 CMC-DNS U PfunXyn / g rye + 0.3 U AniAfur / g rye + 0.3 U CtFe / g rye; Test 5, 1.89 CMC-DNS U PfunXyn / g rye + 0.3 U BaAfur / g rye + 0.3 U CtFe / g rye Test 6, 6.8 pg FveXyn4_1 protein / g rye + 0.3 U AniAfur / g rye + 0.3 U BaAfur / g rye; Test 7, 6.8 pg FveXyn4_1 protein / g rye + 0.3 U AniAfur / g rye + 0.3 U CtFe / g rye; Test 8, 6.8 pg FveXyn4_1 protein / g rye + 0.3 U BaAfur / g rye + 0.3 U CtFe / g rye. In addition to enabling the production of fermentable sugars and appropriate FAN levels, 0.730 mg AMYLEX® 6T / g rye grain meal was added to each test. The grain meal was saccharified using the procedure; heating to 45°C and holding for 40 minutes for starch saccharification to start; heating to 65°C for 10.0 minutes by increasing the temperature by 1 °C / minute; holding at 65°C for 40 minutes; heating to 74°C for 10 minutes by increasing the temperature by 1 °C / minute; holding at 74°C for 160 minutes and allowing the starch saccharification to end.

[0182] Afterwards, when the temperature reached 74°C and the starch saccharification was finished, the iodine test was performed. The time in minutes required to reach iodine negativity was recorded and the results are given in Table 2. It is clear here that a lower dose of the uninhibited FveXyn4_1 was able to obtain iodine negative results for the mash in a shorter time compared to PfunXyn, which indicates that the starch fraction of the rye material was further processed.

[0183] Table 2, Iodine test of rye starch saccharification. The time in minutes required to reach iodine negativity was recorded by OK for tests 1-2 with the following a-amylases added: Test 1, 6.8 pg FveXyn4_1 protein / g rye; Test 2, 1.89 CMC-DNS U PfunXyn / g rye.

[0184]

[0185] After starch liquefaction was finished, each beaker was adjusted to 263 g with tap water and cooled to 25 °C before centrifugation. Separation was performed by centrifugation; in a swing bucket centrifuge (Thermo Scientific SL 8R centrifuge) at 4000 rpm for 5 minutes (5 °C). After centrifugation the volume of the supernatant was measured in mL (volumetric cylinder) and the results are given in Table 3. It is clear that the supernatant volume produced by the uninhibited FveXyn4_1 is much larger, corresponding to an increased filtration yield.

[0186] Table 3, wort supernatant volume after centrifugation. Experiment 1, 6.8 pg FveXyn4_1 protein / g rye; Experiment 2, 1.89 CMC-DNS U PfunXyn / g rye.

[0187]

[0188] According to Dupont Standard Instruction Brewing 23.8580-B28, the turbidity of the wort supernatant samples after starch liquefaction was measured using Anton Paar (DMA 5000, HazeQC ME module) and showed that the turbidity was very different depending on the xylanase used in the experiment. The turbidity was measured at 90° scattering angle (S90 / S25 EBC) to detect the presence of small particles and included the turbidity measured at 25° scattering angle as additional information about larger particles. The turbidity data are shown in Table 4, with S90 and S25 turbidity, respectively. Figure 1 and 2 The three samples: Experiments 3, 4 and 5, all with PfunXyn, all had significantly higher color values than the corresponding samples 6, 7 and 8 with FveXyn4_1. Thus, the color data are in agreement with the observed supernatant volume differences, all samples from the series with PfunXyn produced higher turbidity than the samples from the series with FveXyn4_1, which indicates that FveXyn4_1 effectively reduces the particles in the solution. It was further found that this was independent of the two a-L-arabinofuranosidases and ferulic acid esterases added as ancillary enzymes together with the xylanase. These results clearly demonstrate the superior properties of FveXyn4_1 for rye arabinoxylan breakdown.

[0189] The specific gravity, density and viscosity of the supernatant of the rye wort samples were determined using a DMA 5000 densitometer and a Lovis 2000 M / ME falling ball viscometer (both from Anton Paar, Gratz. Austria).

[0190] The initial extract concentration (OE) after starch saccharification was measured using Anton Paar (DMA 5000) following DuPont standard brew instruction 23.8580-B28, the extract in the sample. The results are shown in Table 4.

[0191] From the data in Table 4, it is observed that not all samples are given data, this is simply due to too high viscosity. The extract content of all samples seems very similar, which means that the application of arabinofuranosidase and / or feruloyl esterase does not cause an increase in extract, e.g. water insoluble extract does not become soluble. The extract content in samples applying PfunXyn or FveXyn4_1 is also similar, samples with FveXyn4_1 as well as arabinofuranosidase and / or feruloyl esterase show a slight tendency to higher extract. The density and specific gravity of all samples are found to be very similar. It is clear from the viscosity data as determined by Anton Paar that the viscosity of the mash is lower when applying FveXyn4_1 compared to samples applying PfunXyn and that the viscosity varies less from sample to sample.

[0192] Table 4, Density (g / cm3), specific gravity (SG), initial extract concentration and viscosity of rye mash supernatant with xylanase, a-L-arabinofuranosidase (Afur) and feruloyl esterase (CtFe). 3

[0193]

[0194] In addition to the characterization of the mash by Anton Paar, the shear (flow) viscosity of the samples was measured using an Anton Paar MCR301 or MCR302 e-type rheometer with measuring system "Double Gap (DG26.7)" at 20 °C. The viscosity was measured using a logarithmic ramp (integration time of 20 to 1 s) over a shear rate of 0.1 to 100 s-1. The viscosity result was obtained at a specific shear rate of 10 s-1 and is shown in Table 5.

[0195] Table 5, Viscosity determined for rye mash supernatant with xylanase, a-L- arabinofuranosidase (Afur) and feruloyl esterase (CtFe). The viscosity was read at a shear rate of 10 s-1 at 20 °C.

[0196]

[0197] ​The viscosity reduction achieved by FveXyn4_1 when used alone was higher than the viscosity reduction achieved when PfunXyn was applied in combination with one or both of the arabinofuranosidases. This indicates that FveXyn4_1 does not need to cleave both mono- and di-substituted arabinofuranose units present in rye arabinoxylan to be able to hydrolyze the xylan backbone of arabinoxylan. This can mean that PfunXyn needs a longer unsubstituted xylan backbone to achieve hydrolysis than FveXyn4_1. In other words, FveXyn4_1 can be able to hydrolyze xylan with a higher degree of substitution of arabinofuranose.

[0198] Example 7 - Application of xylanase for rye starch saccharification in a brewing analysis and determination of real degree of fermentation (RDF) and alcohol content

[0199] A rye mash was prepared similarly to Example 6 with the following differences. Rye grain meal (65.0 g milled rye kernels) was mixed in a beaker with 220 g tap water in a starch saccharification bath (Lockner, LG-Electronics Division) cup and the pH was adjusted to pH 5.5 with 2.5 M sulfuric acid so that the ratio of water to grain meal was 3.38 : 1.

[0200] FveXyn4_1 xylanase variants were added based on mg protein determined according to Example 4 in the following settings:

[0201] Trials 1 to 3, 1.89 CMC-DNS U PfunXyn / g rye; Trials 4 to 6, 6.8 pg FveXyn4_1 protein / g rye. In addition to enabling the production of fermentable sugars and appropriate FAN levels, in each trial, 0.184 mg AMYLEX® 6T / g rye grain meal was added at the start of the starch saccharification at 66°C and 0.184 mg AMYLEX® 6T / g rye grain meal was added at the start of the main starch saccharification at 74°C. The grain meal was starch saccharified using the procedure; heating to 66°C for starch saccharification start and pH adjustment, then applying enzymes and thereafter holding at 66°C for 40 minutes; heating to 74°C by increasing the temperature by 1°C / minute for 8.0 minutes; holding at 74°C for 55 minutes; second application of AMYLEX® 6T and holding at 74°C for another 105 minutes; adjusting the content of each sample to 285 g; cooling to 35°C. 220 g of the various mashes were weighed into 500 mL conical flasks for fermentation, 15 mL of yeast slurry (0.5% yeast, SafSpirit HG-1 dry yeast for distilling from Fermentis) was added to the mash at 35°C; mash fermentation was performed on the grain.

[0202] The remaining wort was separated by centrifugation; in a swing-bucket centrifuge (Thermo Scientific SL 8R centrifuge) at 4000 rpm for 5 minutes (5 °C). The supernatant was used for analysis, see below. After yeast addition, the samples were fermented at 35 °C and 150 rpm. To all samples 0.262 mg DIAZYME® 480 / g rye was added to promote mashing during fermentation. Analysis was performed when fermentation was completed.

[0203] Supernatant analysis: The initial extract concentration (OE) after starch mashing was measured (Lovis) following Standard Brewing Instructions 23.8580-B28, extract in wort samples. The content of free alpha-amino nitrogen (FAN) (mg / litre) was measured in the wort following Standard Brewing Instructions 23.8580-B15, using a spectrophotometer Genesys 10S UV-Vis (based on EBC 8.10).

[0204] Beer analysis: RDF was measured using Anton Paar (DMA 5000) following Standard Brewing Instructions 23.8580-B28 and alcohol was measured by Standard Brewing Instructions 23.8580-B28.

[0205] The Real Fermentation Degree (RDF) value can be calculated according to the following formula:

[0206]

[0207] where: RE = Real Extract Concentration = (0.1808 x °P 初始 ) + (0.8192 x °P 最终 ), °P 初始 is the specific weight of the standardized wort before fermentation and °P 最终 is the specific weight of the fermented wort expressed in Plato degrees.

[0208] In the present context, the Real Fermentation Degree (RDF) is determined from the specific weight and the alcohol concentration.

[0209] The specific weight and the alcohol concentration were determined for the fermented samples using Beer Alcolyzer Plus and DMA 5000 densitometer (both from Anton Paar, Graz, Austria). Based on these measurements, the Real Fermentation Degree (RDF) value was calculated according to the following formula:

[0210]

[0211] where: E(r) is the Real Extract Concentration in Plato degrees (°P) and OE is the Initial Extract Concentration in °P.

[0212] Initial extract concentration (OE) after starch saccharification was measured using Anton Paar (DMA 5000) following Standard Brewing Instructions 23.8580-B28, and the extract in the beer sample. Standard alcohol by volume (% V / V) was measured using Anton Paar (DMA 5000) following Standard Brewing Instructions 23.8580-B28 to determine the achieved standard alcohol by volume (ABV).

[0213] The actual fermentation degree (RDF), extract concentration, and alcohol content of the fermented beers are shown in Table 6. The RDF values, extract concentrations, and alcohol values achieved for the fermented beers applying FveXyn4_1 in the starch saccharification were all higher compared to PfunXyn. The average extract concentration for the three fermentations with PfunXyn was 16.71 °P, while the average extract concentration for the three fermentations with FveXyn4_1 was 17.04 °P. The average RDF for the three fermentations with PfunXyn was 77.50%, while the average RDF for the three fermentations with FveXyn4_1 was 77.58%. The average alcohol content for the three fermentations with PfunXyn was 8.70%, while the average alcohol content for the three fermentations with FveXyn4_1 was 8.89%.

[0214] It is clear that the increased arabinoxylan solubilization during starch saccharification also increased the RDF and alcohol content in the fermented rye wort including FveXyn4_1.

[0215] Table 6. Extract concentration, RDF, and alcohol content of beer fermentations of wort produced with different xylanases.

[0216]

[0217] Example 8 - Arabinoxylan analysis of rye wort samples produced with different xylanases in combination with a-L-arabinofuranosidase and feruloyl esterase.

[0218] The soluble arabinoxylan content of the rye mashes produced as described in Example 6 was quantified. LC-MS reversed phase chromatography was used to characterise and quantify the different soluble arabinoxylans according to DP (from AX1 / monomer up to AX7+) fractionated on an Agilent 1290 BinPump system coupled to a Bruker maXis QTOF4G. Chromatographic separation was performed on an Acquity UPLC® HSS T3 column (100 x 2.1 mm id, 1.8 pm) using a water:methanol:TBA:acetic acid 980:20:2.4:10 (A), methanol:TBA:acetic acid 1000:2.4:10 (B) gradient run for 16 min at an injection volume of 10 mΐ, flow rate of 200 mΐ / min. The effluent from the column was coupled via an electrospray ionisation interface (ionisation: negative mode ESI (electrospray)) to a high resolution maXis mass spectrometer. The following spectrometer settings were applied: nebuliser: 2.0 bar, dry gas: 8 L / min, and dry heater: 200 °C.

[0219] The relative distribution of soluble AX components in rye mashes prepared with xylanase together with a-L-arabinofuranosidase and feruloyl esterase is shown in Table 7. Quantification was performed using HyStar LC with Agilent ICF software. To identify the soluble AX components the following pentose standards were applied: xylose, xylotriose, xylotetraose, xylopentaose, xylohexaose and xyloheptaose. AX fractions were determined for both samples applying PfunXyn or FveXyn4_1 in combination with a-L-arabinofuranosidase and feruloyl esterase. The method was limited to determine soluble arabinoxylans (meaning AX9 or lower).

[0220] The data clearly show that for the sample applying PfunXyn the proportion of AX2 and AX3 is significantly higher and the proportion of AX4 to AX7 is lower. Thus, FveXyn4_1 clearly demonstrates an increased specificity towards the generation of larger soluble dextrins, supporting a more efficient turnover of high molecular AX components and thereby reducing solution viscosity.

[0221] Table 7. Relative distribution of arabinoxylans (AX) according to DP in rye mashes prepared with xylanase together with a-L-arabinofuranosidase and feruloyl esterase.

[0222]

[0223] Example 9 - Xylanase rye and wheat inhibition assay.

[0224] One factor that can limit the efficiency of xylanase action on various cereals can be the presence of specific protein inhibitors of xylanases in the grain. Soluble protein inhibitors of xylanases have been found in barley, rye, corn and other cereals. This example describes a simple and novel sensitivity assay for rye and wheat xylanase inhibitor protein-like (XIP-like) inhibitors. The degree of inhibition of a number of xylanase products by rye and wheat was determined.

[0225] Rye and wheat XIP inhibitor extracts were prepared by the following procedure. A weighed amount (20 g) of rye or wheat flour (Valsemøllen A / S, Denmark) was suspended in 200 mL (10% w / v) of 0.1 M Na-acetate buffer (pH 5.6) and the mixture was stirred on a shaker at 300 rpm for 3.0 h. The insoluble fraction was separated by centrifugation at 5,000 rpm for 20 min in a swing-bucket centrifuge. The supernatant was treated as rye and wheat XIP inhibitor extracts (pH 5.6), respectively. Using 0.1 M Na-acetate buffer (pH 5.6), rye XIP inhibitor extract (pH 5.6) and wheat XIP inhibitor extract (pH 5.6) a dilution series ranging from 500 - 1000000-fold was prepared with the following xylanase samples; BsbBglu + AkaXyl_var, TrXyl, PfunXyn, TemXyn and FveXyn4_1 all obtained from DuPont Nutrition Biosciences ApS, Denmark, Shearzyme P105, Viscoferm and Ultraflo Max obtained from Novozymes A / S, Denmark. All combinations of diluted enzyme samples were incubated at 5°C for 24 hours to promote potential inhibition, subsequently 1 mL 0.1 M Na-acetate buffer (pH 5.6) was mixed with 1 mL diluted enzyme sample in a reaction tube and equilibrated using a water bath at 40°C for 5 min. One piece of Xylazyme (Xylazyme from Megazyme, T-XYZ-200T, for the determination of endo-1,4-b-xylanase activity) purchased from Megazyme (Ireland) was added to each reaction tube, incubated at 40°C for 10 min and all reactions were terminated by the addition of 10 mL demineralized water according to the manufacturer's declaration. The reaction solution was filtered through a paper filter (Whatman, Qualitative filter paper, round, Class 597 Plus, Thermo Fisher Scientific) and the absorbance at 590 nm was determined using a spectrophotometer (Genesys Spectrophotometer 10S UV-Vis). The % rye XIP inhibition and % wheat XIP inhibition was calculated using the following formula, only using the absorbance values in the range of 0.0 to 1.0 produced by the enzyme sample dilutions:

[0226]

[0227] or

[0228]

[0229] The results of the rye and wheat XIP inhibition testing of the various xylanase samples are shown in Table 8. A very large difference was surprisingly found, FveXyn4_1 clearly showed very little XIP inhibition, especially when incubated with rye (17.9% inhibition), however also very little XIP inhibition in the presence of wheat (51.1% inhibition), which can explain the superior viscosity reduction obtained in rye wort with FveXyn4_1. Of all the xylanase samples tested, FveXyn4_1 showed superior low rye XIP inhibition.

[0230] Table 8, xylanase activity (Xylazyme, Megazyme) after incubation for 24 hours at 5°C in 0.1 M Na-acetate buffer (pH 5.6), rye XIP inhibitor extract (pH 5.6) and wheat XIP inhibitor extract (pH 5.6), respectively. Rye XIP inhibition % and wheat XIP inhibition % were calculated according to the formula as described in Example 9.

[0231]

[0232] Example 10 - Rye mashing experiments and dose-response testing of inhibited and uninhibited xylanases and their impact on viscosity reduction and filterability.

[0233] The objective of this example was to test the potential benefits of the presence of uninhibited rye XIP xylanases over inhibited xylanases during a 100% rye based mashing process, using a large dose range. The enzymes were tested in a model system of the starch mashing operation for wort production, using milled rye (supplied by Hiram Walker, USA, material code: 202109-2201) (milled on a Buhler-Miag mill (0.5 mm setting, position 4)). As an example of a rye XIP uninhibited xylanase, FveXyn4_1 was applied (17.9% inhibition), according to the rye XIP inhibition as described in Example 9, while as an example of a rye XIP inhibited xylanase, PfunXyn was applied (84.9% inhibition).

[0234] Starch saccharification procedure for wort production

[0235] Rye meal (60.0 g milled rye grain) was mixed in a beaker with 203 g tap water in a starch liquefaction bath (Lockner, LG-Electronics Division) cup and the pH was adjusted to pH 5.5 with 2.5 M sulfuric acid, such that the ratio of water to rye meal was 3.38 : 1. FveXyn4_1 xylanase variant was added based on mg protein determined according to Example 4. PfunXyn was applied at 0.787 to 15.7 CMC-DNS U / g rye and FveXyn4_1 for comparison was applied at 5.7 to 113.3 pg FveXyn4_1 protein / g rye. In all trials, 0.368 mg AMYLEX® 6T / g rye meal was added in the starch liquefaction at 66°C to enable fermentable sugar and proper FAN levels to be generated. The meal was starch liquefied using the program; heated to 45°C for starch liquefaction start and pH adjustment, then enzyme was applied and thereafter held at 45°C for 40 minutes; heated to 65°C by increasing the temperature by 1°C / minute for 20 minutes; held at 65°C for 40 minutes; heated to 74°C by increasing the temperature by 1°C / minute for 9 minutes; and held at 74°C for another 160 minutes; the content of each sample was adjusted to 263 g; cooled to 25°C, then centrifuged.

[0236] Wort separation was performed by centrifugation; in a swing bucket centrifuge (Thermo Scientific SL 8R centrifuge) at 4000 rpm for 5 minutes (5°C). The volume of wort was measured in mL after centrifugation (graduated cylinder). The wort samples were boiled in a boiling bath for 60 minutes and the samples were cooled to 17°C. All samples were filtered and used for analysis, see below.

[0237] The wort sample extract concentration, density and flow viscosity was determined as described in Example 6, the results are shown in Tables 9 and 10.

[0238] The density and specific gravity of all samples were found to be very similar. A slight increase in initial extract concentration was observed by increasing the dosage of both xylanase samples.

[0239] Surprisingly, it was evident from the determined viscosity data that the viscosity of the wort was lower when FveXyn4_1 was applied in a large dosage range compared to the samples where PfunXyn was applied, and the viscosity varied less from sample to sample. All dosages of FveXyn4_1 resulted in lower wort viscosity compared to any dosage of PfunXyn. In addition, as seen in Table 9, the volume of supernatant collected from the wort treatment was significantly larger for all dosages of FveXyn4_1 compared to PfunXyn, enabling a higher yield.

[0240] Table 9. Wortsample density (g / cm3), specific gravity (SG), initial extract concentration and flow viscosity (read at 20 °C at shear rate 10 s1) determined for rye mashes with various doses of xylanases PfunXyn and FveXyn4_1 as indicated. 3

[0241]

[0242] Table 10. Volume collected after centrifugation of rye mashes with various doses of xylanases PfunXyn and FveXyn4_1 as indicated.

[0243]

[0244] Example 11 - Malt starch saccharification trial and dose-response testing with rye-inhibited and uninhibited xylanases and their impact on viscosity reduction and filterability.

[0245] The purpose of this example was to test uninhibited xylanases in comparison to inhibited xylanases present during a 100% malt-based starch saccharification process using various doses. The enzymes were tested in a model system for starch saccharification operation for wort production using malt ground on a Buhler-Miag malt mill (0.5 mm setting, position 4) (Pilsner malt from Fuglsang, Denmark, batch no. 12.10.2019). FveXyn4_1 (17.9% inhibition) was applied as an example of a rye XIP uninhibited xylanase and PfunXyn (84.9% inhibition) as an example of a rye XIP inhibited xylanase according to rye XIP inhibition as described in Example 9.

[0246] Starch saccharification procedure for wort production

[0247] Malt grain meal (60.0 g milled malt grains) was mixed in a beaker with 203 g tap water in a starch saccharification bath (Lockner, LG-Electronics Division) cup and the pH was adjusted to pH 5.5 with 2.5 M sulfuric acid so that the ratio of water to grain meal was 3.38 : 1. FveXyn4_1 xylanase variant was added based on mg protein determined according to Example 4. PfunXyn was applied at 0.315 to 1.570 CMC-DNS U / g rye and FveXyn4_1 for comparison at 2.27 to 11.3 pg FveXyn4_1 protein / g malt. The grain meal was saccharified as described in Example 10 and wort separation and subsequent wort analysis was performed. ​

[0248] Mash sample wort extract concentration, density and flow viscosity were determined as described in Example 6, the results are shown in Tables 11 and 12.

[0249] It was found that the density, specific gravity and initial extract concentration of all samples generated by using the two xylanase samples were very similar. In addition, no significant difference in mash viscosity was observed for the wort mash compared to the rye. As seen in Table 12, the volume of supernatant collected from the mash treatment was identical for all doses of FveXyn4_1 and PfunXyn applied, thereby indicating that the application in the wort mash was not affected by the rye XIP-inhibited xylanase compared to the inhibited xylanase.

[0250] Table 11. Worts sample density (g / cm3), specific gravity (SG), initial extract concentration and flow viscosity (read at 20 °C at shear rate 10 s-1) determined for wort mash with various doses of xylanases PfunXyn and FveXyn4_1 as indicated. 3

[0251]

[0252] Table 12. Volume collected after centrifugation of wort mash with various doses of xylanases PfunXyn and FveXyn4_1 as indicated.

[0253]

[0254] Example 12 - Performance of xylanases in a malt starch saccharification test including high rye

[0255] The purpose of this example was to test the presence of rye XIP-inhibited xylanases compared to inhibited xylanases during a starch saccharification process based on 50% malt - 50% rye using various xylanase doses. The enzymes were tested in a model system for starch saccharification operation for wort production using malt (Pilsen malt from Sophus Fuglsang A / S, Denmark, batch no. 10.12.2019) milled on a Buhler-Miag malt mill (0.5 mm setting, position 4) and rye (Wey rye 3-8 EBC from Maltbazaren, Denmark, batch: 12.03.2020, DK20-00066) milled on a Buhler-Miag malt mill (0.5 mm setting, position 4).

[0256] ​As an example of a rye XIP-inhibited xylanase, FveXyn4_1 (17.9% inhibition) was applied, and as an example of a rye XIP-uninhibited xylanase, PfunXyn (84.9% inhibition) and TemXyn (40.0% inhibition) were applied, according to rye XIP inhibition as described in Example 9.

[0257] Starch saccharification procedure for wort production

[0258] Rye grain mash (35.0 g milled rye grain) and malt grain mash (35.0 g milled malt grain) were mixed in a beaker with 210 g tap water in a starch liquefaction bath (Lockner, LG-Electronics Division) cup and the pH was adjusted to pH 5.5 with 2.5 M sulfuric acid, so that the ratio of water to grain mash was 3.1 : 1. FveXyn4_1 xylanase variant was added based on mg protein determined according to Example 4. PfunXyn was applied at 3.15 to 9.45 CMC-DNS U / g grain mash, TemXyn was applied at 5.52 to 16.57 U / g grain mash, and FveXyn4_1 was applied for comparison at 22.7 to 68.0 pg FveXyn4_1 protein / g grain mash. The grain mash was subjected to starch liquefaction using the program; heating to 52°C for starch liquefaction start and pH adjustment, then enzyme was applied, and thereafter held at 52°C for 10 minutes; heating to 65°C for 13 minutes by increasing the temperature by 1°C / minute; held at 65°C for 45 minutes; heating to 72°C for 7 minutes by increasing the temperature by 1°C / minute; and held at 72°C for another 15 minutes; heating to 78°C for 6 minutes by increasing the temperature by 1°C / minute; and held at 78°C for another 30 minutes - starch liquefaction end; the content of each sample was adjusted to 350 g and filtered. The wort volume collected after 5, 10, 15, and 30 minutes was measured and the pH was adjusted to pH 5.2 with 2.5 M sulfuric acid. One hop was added to each flask. The wort samples were boiled in a boiling bath for 60 minutes and the samples were cooled to 17°C. All samples were filtered for analysis, see below.

[0259] Wort sample extract concentration, density, and flow viscosity was determined as described in Example 6, the results are shown in Tables 13 and 14.

[0260] The density and specific gravity of all samples were found to be very similar. A slight increase in actual extract concentration was observed in some samples by the FveXyn4_1 xylanase.

[0261] Surprisingly, it is evident from the determined viscosity data that the mash viscosity is lower and the viscosity variation from sample to sample is less when FveXyn4_1 is applied over a large dose range compared to samples where PfunXyn and TemXyn are applied. FveXyn4_1 at all doses results in lower mash viscosity compared to PfunXyn and TemXyn at any dose applied. In addition, as seen in Table 14, the turbidity (determined as color by EBC scattering at 20°C by 25 and 90 degrees) of the malt-rye wort samples clearly indicates that the malt wort samples prepared with FveXyn4_1 produce very clear samples with lower turbidity or color compared to malt wort prepared with PfunXyn or TemXyn, respectively.

[0262] In addition, as seen in Table 15, the volume of supernatant collected from mash filtration is significantly greater for all doses of FveXyn4_1 compared to all doses of PfunXyn and TemXyn applied, thereby enabling significantly higher yield in processing 50% malt-50% rye mash.

[0263] Table 13, Malt wort sample density (g / cm3), specific gravity (SG), real extract concentration and Anton Paar viscosity (12°P) determined for malt-rye mash with various doses of xylanases PfunXyn, TemXyn and FveXyn4_1 as indicated. 3

[0264]

[0265] Table 14, Turbidity (determined as color by EBC scattering at 20°C by 25 degrees (S25 / S0) or 90 degrees (S90 / S0)) of malt-rye wort samples of mash with various doses of xylanases PfunXyn, TemXyn and FveXyn4_1 as indicated.

[0266]

[0267] Table 15, Filtration volume collected after 5, 10, 15 and 30 min of mash filtration (paper filter) of 50% malt-50% rye mash with various doses of xylanases: PfunXyn, TemXyn and FveXyn4_1 as indicated.

[0268]

[0269] Example 13 - Comparison of xylanase performance in a malt starch saccharification test including high rye, wheat and barley

[0270] ​The purpose of this example was to test various doses of xylanase enzymes on rye XIP-inhibited xylanases present during the starch liquefaction process based on 60% malt : 40% rye, 60% malt : 40% wheat, and 60% malt : 40% barley. The enzymes were tested in a model system for starch liquefaction operation for wort production using malt (Pilsner malt from Sophus Fuglsang A / S, Denmark, batch no. 10.12.2019) milled on a Buhler-Miag malt mill (0.5 mm setting, position 4), milled rye (Wey rye 3-8 EBC from Maltbazaren, Denmark, batch: 12.03.2020, DK20-00066) milled on a Buhler-Miag malt mill (0.5 mm setting, position 4), barley (green barley from Sophus Fuglsang A / S, Denmark, batch no. 09.03.2019) milled on a Buhler-Miag malt mill (0.5 mm setting, position 4), and wheat (organic whole grain wheat from XXX, batch no. 17.11.2022-1) milled on a Buhler-Miag malt mill (0.5 mm setting, position 4).

[0271] According to the rye XIP inhibition as described in Example 9, FveXyn4_1 (17.9% inhibition) was applied as an example of a rye XIP-inhibited xylanase, and TemXyn (40.0% inhibition) was applied as an example of a rye XIP-uninhibited xylanase.

[0272] Starch saccharification procedure for wort production

[0273] Rye, wheat, or barley grist (28.0 g milled rye kernels) and malt grist (42.0 g milled malt kernels) were mixed in a beaker with 210 g tap water in a starch liquefaction bath (Lockner, LG-Electronics Division) cup and the pH was adjusted to pH 5.5 with 2.5 M sulfuric acid so that the ratio of water to grist was 3.1 : 1. FveXyn4_1 xylanase variants were added based on mg protein determined according to Example 4. TemXyn was applied at 0.27 to 0.69 U / g grist and FveXyn4_1 for comparison was applied at 9.0 to 27.2 pg FveXyn4_1 protein / g grist. BsdBglu was added to all trials to support the xylanases at a varying dose of 2-5 U / kg grist, which was the same for both xylanases used.

[0274] The grain milled material was saccharified using the procedure; heated to 60 °C for starch saccharification start and pH adjustment, then enzyme was applied, and thereafter held at 60 °C for 5 minutes; heated to 65 °C for 5 minutes by increasing the temperature by 1 °C / minute; held at 65 °C for 45 minutes; heated to 72 °C for 7 minutes by increasing the temperature by 1 °C / minute; and held at 72 °C for another 20 minutes; heated to 78 °C for 6 minutes by increasing the temperature by 1 °C / minute; and held at 78 °C for another 10 minutes - starch saccharification end; the content of each sample was adjusted to 350 g and filtered. The wort volume collected after 5, 10, 15, and 30 minutes was measured (in ml) Figure 3 The results are shown as the average of each xylanase at all doses (TemXyn was applied at 0.27 to 0.69 U / g grain milled material, and FveXyn4_1 was applied at 9.0 to 27.2 pg FveXyn4_1 protein / g grain milled material).

[0275] The wort sample extract concentration and viscosity was determined as described in Example 6, Figure 4 and 5 The results are shown as the average of each xylanase at all doses (TemXyn was applied at 0.27 to 0.69 U / g grain milled material, and FveXyn4_1 was applied at 9.0 to 27.2 pg FveXyn4_1 protein / g grain milled material).

[0276] It can be observed that the average wort volume collected after 30 minutes of filtration increased using FveXyn4_1 compared to TemXyn (in ml) Figure 3 ) over the xylanase dose range. Furthermore, it is evident that the relative increase was highest for the mash consisting of 40 : 60 rye : malt, followed by the mash consisting of 40 : 60 wheat : malt, and the relative increase was smallest for the mash consisting of 40 : 60 barley : malt.

[0277] It can be seen from Figure 4 that the average wort viscosity at 12 °P (mPa x s) decreased using FveXyn4_1 compared to TemXyn (in ml) over the xylanase dose range. The relative decrease was observed to be largest for the mash consisting of 40 : 60 rye : malt, followed by the mash consisting of 40 : 60 wheat : malt, and the relative decrease was smallest for the mash consisting of 40 : 60 barley : malt.

[0278] It can be seen from Figure 5It can be seen that, within the xylanase dose range, the average initial wort concentration (°P) after filtration is increased using FveXyn4_1 compared to TemXyn. The relative increase is largest for the 40 : 60 rye : malt mash and for the 40 : 60 wheat : malt mash, while the 40 : 60 barley : malt mash only increases slightly.

[0279] The benefit of using the non-rye XIP-inhibited xylanase FveXyn4_1 in terms of increasing filtration volume or rate by reducing viscosity and increasing initial wort concentration (°P) is clearly most pronounced when using rye or wheat in the mash compared to malt or barley, which corresponds to the lower XIP inhibition of these raw materials.

[0280] Example 14 - Effect of xylanases on ethanol production in rye-malt SSF trials including high rye.

[0281] The purpose of this example was to test the non-rye XIP-inhibited xylanases in comparison to the inhibited xylanases present during a rye-based starch saccharification and fermentation process using a variety of xylanase doses. The enzymes were tested in a model system for starch saccharification operation for wort production using malt (brewers malt, DK 19-01017, Denmark) milled on a Buhler-Miag malt mill (0.5 mm setting, position 4) and milled rye (Briess raw rye: Denmark, batch: DK 20-00053) milled on a Buhler-Miag malt mill (0.5 mm setting, position 4). FveXyn4_1 (17.9% inhibition) was applied as an example of a non-rye XIP-inhibited xylanase and PfunXyn (84.9% inhibition) and Viscoferm (41.3% inhibition) were applied as examples of rye XIP-inhibited xylanases according to the rye XIP inhibition as described in Example 9.

[0282] Mash protocol without enzyme addition

[0283] All raw materials were milled at setting number 4 and mixed with water, where the mash temperature was 55 °C. The adjunct liquefaction: The temperature was adjusted to 55 °C for 1 minute for the start of the starch saccharification, 66.5 g rye + 1.75 g malt total cereal grind was added to 210.0 g water. The pH was adjusted to 5.5 if needed. No enzymes were added. The temperature was increased to 95 °C at 1 °C / minute and held for 40 minutes. The saccharification was tested at 10, 20 and 30 min at 95 °C with iodine tests.

[0284] Main starch liquefaction: The temperature of the starch liquefaction was adjusted to 65°C. Therefore, the mash was cooled to 65°C and 1.75 g malt was added during the conversion. The mash was kept at 65°C for 30 minutes and after 30 minutes the starch was tested for saccharification with iodine. Then it was cooled to 32°C for 5 minutes and each beaker was adjusted to 280 g with tap water. The mash was filled into 500 ml Erlenmeyer flasks for fermentation, 0.5% yeast was added to the wort at 32°C / 90°F (280 g mash 1.4 g) (White Labs Distillery Yeast: DK 202009-0301). Then the yeast (White Labs Distillery Yeast: DK 202009-0301) was added for fermentation. No enzymes were added and the mash was fermented on the grain. The temperature was set to 35°C / 95°F and the fermentation lasted for 3 days.

[0285] Mash protocol with enzyme addition

[0286] Adjunct liquefaction: The temperature was adjusted to 55°C for 1 minute for the start of starch saccharification, 66.5 g rye + 1.75 g malt total grain comminution was added to 210.0 grams of water. If necessary, the pH was adjusted to 5.5. After measuring the correct pH, alpha amylase (0.15 kg / t grain comminution) was added to ensure efficient liquefaction (AMYLEX 5T, 1.050 g dissolved in 100 mL, provided by DuPont Nutrition Biosciences A / S, Denmark). The temperature was increased to 95°C at 1 °C / minute and held for 40 minutes. The saccharification was tested at 95°C at 10, 20 and 30 min with iodine testing.

[0287] Main starch liquefaction: The temperature of the starch liquefaction was adjusted to 65°C. Xylanase was applied in g sample / kg grain comminution according to table 16 below. Therefore, the mash was cooled to 65°C and 1.75 g malt was added during the conversion. The mash was kept at 65°C for 30 minutes and after 30 minutes the starch was tested for saccharification with iodine. Then it was cooled to 32°C for 5 minutes and each beaker was adjusted to 280 g with tap water.

[0288] The mash was filled into 500 ml Erlenmeyer flasks for fermentation, 0.5% yeast was added to the wort at 32°C / 90°F (280 g mash 1.4 g) (White Labs Distillery Yeast: DK 202009-0301). For all trials, glucoamylase (0.8 kg / t grain comminution) was added to ensure efficient saccharification (Diazyme SSF2, 1.750 g dissolved in 100 mL, provided by DuPont Nutrition Biosciences A / S, Denmark). The mash was fermented on the grain. The temperature was set to 35°C / 95°F and the fermentation lasted for 3 days.

[0289] Table 16, test setup for addition of raw material including addition of xylanases (Viscoferm, PfunXyn and FveXyn4_1) in liquefaction at 95°C and main starch saccharification at 65°C.

[0290]

[0291] The wort sample density, specific gravity and viscosity were determined as described in Example 6, the results of which are shown in Table 17. The density and specific gravity of all samples were found to be very similar, the viscosity of the control was too high to be analysed.

[0292] Again, it is clear from the determined viscosity data that the mash viscosity is lower and the variation in viscosity from sample to sample is less when FveXyn4_1 is applied over a large dose range compared to the samples in which PfunXyn and Viscoferm are applied. FveXyn4_1 at all doses results in a lower mash viscosity compared to PfunXyn and TemXyn at any dose applied.

[0293] Table 17, wort sample density (g / cm3), specific gravity (SG), real extract concentration and Anton Paar viscosity (12°P) determined for malt-rye mashes with various doses of xylanases Viscoferm, PfunXyn and FveXyn4_1 as indicated.

[0294]

[0295] The real degree of fermentation was measured using Anton Paar (DMA 5000) following Standard Brewing Instructions 23.8580-B28 and the standard alcohol degree (% V / V) (ABV) was measured using Anton Paar (DMA 5000) following Standard Brewing Instructions 23.8580-B28.

[0296] The effect of increasing the rate of application of xylanase on the ethanol yield was also observed. In addition to the lowest viscosity, FveXyn4_1 also provided the highest level of ethanol, as shown in Figure 6 The results clearly show that increasing the rate of application of FveXyn4_1 also results in an increase in ethanol yield and more than that obtained by PfunXyn and FveXyn4_1 in a rye-based SSF fermentation.

Claims

1. A method for producing rye whiskey, the method comprising the steps of: (a) providing a cereal mash comprising 51 w / w% rye grain; (b) adding water to the cereal mash to provide a mash; (c) subjecting the mash of step (b) to pre-liquefaction; (d) gelatinizing the mash of step (c); (e) subjecting the mash of step (d) to liquefaction in the presence of a xylanase having reduced sensitivity to rye XIP inhibitors; (f) subjecting the mash of step (e) to saccharification; (g) fermenting the mash of step (f) to produce a fermentation containing ethanol; (h) distilling the fermentation of step (g) to provide the rye whiskey.

2. The method of claim 1, wherein the xylanase is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% inhibited by rye XIP inhibitors.

3. The method of claim 2, wherein the xylanase is less than 20% inhibited by rye XIP inhibitors.

4. The method of any of the preceding claims, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof.

5. The method of claim 4, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof.

6. The method of claim 5, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof.

7. The method of claim 6, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof.

8. The method of claim 7, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof.

9. The method of claim 8, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof.

10. The method of claim 9, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

11. The method of claim 10, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

12. The method of claim 11, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide according to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

13. The method of claim 12, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide according to SEQ ID NO: 2 or SEQ ID NO:

3.

14. The method of any one of the preceding claims, wherein the rye kernels are malted.

15. The method of any one of claims 1 to 13, wherein the rye kernels are unmalted.

16. The method of any one of claims 1 to 15, wherein the rye kernels are milled.

17. The method of any one of the preceding claims, wherein the grain comminution is 60, 70, 80, 85, 90, 95, 99, or 100 w / w% rye.

18. The method of any one of the preceding claims, wherein the grain comminution further comprises malted or unmalted barley, wheat, corn, rye, rice, cassava, oat flakes, or sorghum.

19. The method of claim 18, wherein the grain comminution further comprises malted barley.

20. The method of any one of the preceding claims, further comprising adding a rye- inhibited xylanase to any one of steps (b), (c), and / or (d).

21. The method of any one of the preceding claims, further comprising adding an alpha- amylase and / or a beta-glucanase to any one of steps (b), (c), (d), and / or (e).

22. The method of claim 21, wherein a beta-glucanase is added.

23. The method of claim 22, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof.

24. The method of claim 23, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 80% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof.

25. The method of claim 24, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 85% sequence identity to SEQ ID NO: 8 or a beta-glucanase-activity fragment thereof.

26. The method of claim 25, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 90% sequence identity to SEQ ID NO: 8 or a beta-glucanase-activity fragment thereof.

27. The method of claim 26, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 92% sequence identity to SEQ ID NO: 8 or a beta-glucanase-activity fragment thereof.

28. The method of claim 27, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 8 or a beta-glucanase-activity fragment thereof.

29. The method of claim 28, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 98% sequence identity to SEQ ID NO: 8 or a beta-glucanase-activity fragment thereof.

30. The method of claim 29, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NO: 8 or a beta-glucanase-activity fragment thereof.

31. The method of claim 30, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide according to SEQ ID NO: 8 or a beta- glucanase-activity fragment thereof.

32. The method of claim 31, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide according to SEQ ID NO:

8.

33. The method of any of the preceding claims, further comprising adding a glucoamylase in step (f).

34. The method of any of the preceding claims, further comprising adding a protease in step (g).

35. The method of any of the preceding claims, further comprising adding an alpha-L-arabinofuranosidase in any of steps (b), (c), (d), and / or (e).

36. The method of claim 35, wherein the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase- activity fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase- activity fragment thereof.

37. The method of claim 36, wherein the a-L-arabinofuranosidase is an enzyme having a-L-arabinofuranosidase activity comprising a polypeptide having at least 80% sequence identity to SEQ ID NO: 10 or an a-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an a-L-arabinofuranosidase active fragment thereof.

38. The method of claim 37, wherein the a-L-arabinofuranosidase is an enzyme having a-L-arabinofuranosidase activity comprising a polypeptide having at least 85% sequence identity to SEQ ID NO: 10 or an a-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an a-L-arabinofuranosidase active fragment thereof.

39. The method of claim 38, wherein the a-L-arabinofuranosidase is an enzyme having a-L-arabinofuranosidase activity comprising a polypeptide having at least 90% sequence identity to SEQ ID NO: 10 or an a-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an a-L-arabinofuranosidase active fragment thereof.

40. The method of claim 39, wherein the a-L-arabinofuranosidase is an enzyme having a-L-arabinofuranosidase activity comprising a polypeptide having at least 92% sequence identity to SEQ ID NO: 10 or an a-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an a-L-arabinofuranosidase active fragment thereof.

41. The method of claim 40, wherein the a-L-arabinofuranosidase is an enzyme having a-L-arabinofuranosidase activity comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 10 or an a-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an a-L-arabinofuranosidase active fragment thereof.

42. The method of claim 41, wherein the a-L-arabinofuranosidase is an enzyme having a-L-arabinofuranosidase activity comprising a polypeptide having at least 98% sequence identity to SEQ ID NO: 10 or an a-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an a-L-arabinofuranosidase active fragment thereof.

43. The method of claim 42, wherein the a-L-arabinofuranosidase is an enzyme having a-L-arabinofuranosidase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NO: 10 or an a-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an a-L-arabinofuranosidase active fragment thereof.

44. The method of claim 43, wherein the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity comprising a polypeptide according to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.

45. The method of any of the preceding claims, further comprising adding ferulic acid esterase in any of steps (b), (c), (d) and / or (e).

46. The method of claim 45, wherein the ferulic acid esterase is an enzyme having ferulic acid esterase activity comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 12 or a ferulic acid esterase active fragment thereof.

47. The method of claim 46, wherein the ferulic acid esterase is an enzyme having ferulic acid esterase activity comprising a polypeptide having at least 80% sequence identity to SEQ ID NO: 12 or a ferulic acid esterase active fragment thereof.

48. The method of claim 47, wherein the ferulic acid esterase is an enzyme having ferulic acid esterase activity comprising a polypeptide having at least 85% sequence identity to SEQ ID NO: 12 or a ferulic acid esterase active fragment thereof.

49. The method of claim 48, wherein the ferulic acid esterase is an enzyme having ferulic acid esterase activity comprising a polypeptide having at least 90% sequence identity to SEQ ID NO: 12 or a ferulic acid esterase active fragment thereof.

50. The method of claim 49, wherein the ferulic acid esterase is an enzyme having ferulic acid esterase activity comprising a polypeptide having at least 92% sequence identity to SEQ ID NO: 12 or a ferulic acid esterase active fragment thereof.

51. The method of claim 50, wherein the ferulic acid esterase is an enzyme having ferulic acid esterase activity comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 12 or a ferulic acid esterase active fragment thereof.

52. The method of claim 51, wherein the ferulic acid esterase is an enzyme having ferulic acid esterase activity comprising a polypeptide having at least 98% sequence identity to SEQ ID NO: 12 or a ferulic acid esterase active fragment thereof.

53. The method of claim 52, wherein the ferulic acid esterase is an enzyme having ferulic acid esterase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NO: 12 or a ferulic acid esterase active fragment thereof.

54. The method of claim 53, wherein the ferulic acid esterase is an enzyme having ferulic acid esterase activity comprising a polypeptide according to SEQ ID NO: 12 or a ferulic acid esterase active fragment thereof.

55. A method for preparing a low viscosity mash, the method comprising the steps of: (a) preparing a mash from a cereal grind comprising rye grain in the presence of a xylanase having reduced sensitivity to rye XIP inhibitors; (b) optionally filtering the mash to obtain wort.

56. The method of claim 55, wherein the xylanase is less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% inhibited by the rye XIP inhibitor.

57. The method of claim 56, wherein the xylanase is less than 20% inhibited by the rye XIP inhibitor.

58. The method of any one of claims 55-57, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

59. The method of claim 58, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

60. The method of claim 59, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

61. The method of claim 60, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

62. The method of claim 61, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

63. The method of claim 62, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

64. The method of claim 63, wherein the rye-inhibitor-insensitive xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

65. The method of claim 64, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

66. The method of claim 65, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide according to SEQ ID NO: 2 or a xylanase- active fragment thereof, or SEQ ID NO: 3 or a xylanase-active fragment thereof.

67. The method of claim 66, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide according to SEQ ID NO: 2 or SEQ ID NO:

3.

68. The method of any one of claims 55 to 67, wherein the rye kernels are malted.

69. The method of any one of claims 55 to 67, wherein the rye kernels are unmalted.

70. The method of any one of claims 55 to 69, wherein the rye kernels are milled.

71. The method of any one of claims 55 to 70, wherein the grain comminution is 5, 10, 20, 25, 30, 35, 40, 45, 50, 51, 60, 70, 80, 85, 90, 95, 99, or 100 w / w% rye.

72. The method of any one of claims 55 to 71, wherein the grain comminution further comprises malted or unmalted barley, corn, rye, wheat, cassava, oat flakes, or sorghum.

73. The method of claim 72, wherein the grain comminution further comprises malted barley.

74. The method of any one of claims 55 to 73, further comprising adding an alpha-amylase and / or a beta-glucanase to the mash.

75. The method of claim 74, wherein a beta-glucanase is added.

76. The method of claim 75, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof.

77. The method of claim 76, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 80% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof.

78. The method of claim 77, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 85% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof.

79. The method of claim 78, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 90% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof.

80. The method of claim 79, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 92% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof.

81. The method of claim 80, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 8 or a beta-glucanase-activity fragment thereof.

82. The method of claim 81, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 98% sequence identity to SEQ ID NO: 8 or a beta-glucanase-activity fragment thereof.

83. The method of claim 82, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NO: 8 or a beta-glucanase-activity fragment thereof.

84. The method of claim 83, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide according to SEQ ID NO: 8 or a beta- glucanase-activity fragment thereof.

85. The method of claim 84, wherein the beta-glucanase is an enzyme having beta- glucanase activity comprising a polypeptide according to SEQ ID NO:

8.

86. The method of any one of claims 55 to 85, further comprising adding an alpha-L- arabinofuranosidase in step (a) and / or (b).

87. The method of claim 85, wherein the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase- activity fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase- activity fragment thereof.

88. The method of claim 87, wherein the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity comprising a polypeptide having at least 80% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase- activity fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase- activity fragment thereof.

89. The method of claim 88, wherein the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity comprising a polypeptide having at least 85% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase- activity fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase- activity fragment thereof.

90. The method of claim 89, wherein the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity comprising a polypeptide having at least 90% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase- activity fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase- activity fragment thereof.

91. The method of claim 90, wherein the a-L-arabinofuranosidase is an enzyme having a-L-arabinofuranosidase activity comprising a polypeptide having at least 92% sequence identity to SEQ ID NO: 10 or an a-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an a-L-arabinofuranosidase active fragment thereof.

92. The method of claim 91, wherein the a-L-arabinofuranosidase is an enzyme having a-L-arabinofuranosidase activity comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 10 or an a-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an a-L-arabinofuranosidase active fragment thereof.

93. The method of claim 92, wherein the a-L-arabinofuranosidase is an enzyme having a-L-arabinofuranosidase activity comprising a polypeptide having at least 98% sequence identity to SEQ ID NO: 10 or an a-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an a-L-arabinofuranosidase active fragment thereof.

94. The method of claim 93, wherein the a-L-arabinofuranosidase is an enzyme having a-L-arabinofuranosidase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NO: 10 or an a-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an a-L-arabinofuranosidase active fragment thereof.

95. The method of claim 94, wherein the a-L-arabinofuranosidase is an enzyme having a-L-arabinofuranosidase activity comprising a polypeptide according to SEQ ID NO: 10 or an a-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an a-L-arabinofuranosidase active fragment thereof.

96. The method of any one of claims 55 to 95, further comprising adding a feruloyl esterase in step (a) and / or (b).

97. The method of claim 96, wherein the feruloyl esterase is an enzyme having feruloyl esterase activity comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

98. The method of claim 97, wherein the feruloyl esterase is an enzyme having feruloyl esterase activity comprising a polypeptide having at least 80% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

99. The method of claim 98, wherein the feruloyl esterase is an enzyme having feruloyl esterase activity comprising a polypeptide having at least 85% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

100. The method of claim 99, wherein the feruloyl esterase is an enzyme having feruloyl esterase activity comprising a polypeptide having at least 90% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

101. The method of claim 100, wherein the feruloyl esterase is an enzyme having feruloyl esterase activity comprising a polypeptide having at least 92% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

102. The method of claim 101, wherein the feruloyl esterase is an enzyme having feruloyl esterase activity comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

103. The method of claim 102, wherein the feruloyl esterase is an enzyme having feruloyl esterase activity comprising a polypeptide having at least 98% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

104. The method of claim 103, wherein the feruloyl esterase is an enzyme having feruloyl esterase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

105. The method of claim 104, wherein the feruloyl esterase is an enzyme having feruloyl esterase activity comprising a polypeptide according to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

106. A method for identifying a xylanase having reduced sensitivity to a cereal XIP inhibitor, the method comprising assaying the activity of the xylanase in the presence and absence of the cereal XIP inhibitor, and determining the percent inhibition of the cereal XIP inhibitor.

107. The method of claim 106, wherein the cereal is barley, wheat, corn, rye, rice, cassava, oat flakes, or sorghum.

108. The method of claim 106, wherein the cereal is rye or wheat.

109. The method of claim 108, wherein the cereal is rye.

110. The method of claim 108, wherein the cereal is wheat.

111. A xylanase having reduced sensitivity to a rye XIP inhibitor identified by the method of claim 109.

112. The xylanase of claim 111, which is inhibited by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% by a rye XIP inhibitor.

113. The xylanase of claim 112, which is inhibited by less than 20% by a rye XIP inhibitor.

114. The xylanase of claim 113, which comprises an enzyme having xylanase activity comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof.

115. The xylanase of claim 114, comprising an enzyme having xylanase activity comprising a polypeptide having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

116. The xylanase of claim 115, comprising an enzyme having xylanase activity comprising a polypeptide having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

117. The xylanase of claim 116, comprising an enzyme having xylanase activity comprising a polypeptide having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

118. The xylanase of claim 117, comprising an enzyme having xylanase activity comprising a polypeptide having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

119. The xylanase of claim 118, comprising an enzyme having xylanase activity comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

120. The xylanase of claim 119, comprising an enzyme having xylanase activity comprising a polypeptide having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

121. The xylanase of claim 120, comprising an enzyme having xylanase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

122. The xylanase of claim 121, comprising an enzyme having xylanase activity comprising a polypeptide according to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

123. The xylanase of claim 122, comprising an enzyme having xylanase activity comprising a polypeptide according to SEQ ID NO: 2 or SEQ ID NO:

3.

124. A xylanase having reduced sensitivity to wheat XIP inhibitors identified by the method of claim 110.

125. The xylanase of claim 124, which is inhibited by less than 90%, 80%, 70%, 60%, 55%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% by a wheat XIP inhibitor.

126. The xylanase of claim 125, which is inhibited by less than 55% by a wheat XIP inhibitor.

127. The xylanase of claim 126, which comprises an enzyme having xylanase activity comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

128. The xylanase of claim 127, which comprises an enzyme having xylanase activity comprising a polypeptide having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

129. The xylanase of claim 128, which comprises an enzyme having xylanase activity comprising a polypeptide having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

130. The xylanase of claim 129, which comprises an enzyme having xylanase activity comprising a polypeptide having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

131. The xylanase of claim 130, which comprises an enzyme having xylanase activity comprising a polypeptide having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

132. The xylanase of claim 131, which comprises an enzyme having xylanase activity comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

133. The xylanase of claim 132, which comprises an enzyme having xylanase activity comprising a polypeptide having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

134. The xylanase of claim 133, which comprises an enzyme having xylanase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof.

135. The xylanase of claim 134, comprising an enzyme having xylanase activity comprising a polypeptide according to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof.

136. The xylanase of claim 135, comprising an enzyme having xylanase activity comprising a polypeptide according to SEQ ID NO: 2 or SEQ ID NO:

3.

137. A composition for reducing wort viscosity in wheat adjunct brewing, the composition comprising a beta-glucanase and a xylanase having reduced sensitivity to wheat XIP inhibitors.

138. The composition of claim 137, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.

139. The composition of claim 138, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 80% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity comprising a polypeptide having at least 80% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.

140. The composition of claim 139, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 85% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity comprising a polypeptide having at least 85% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.

141. The composition of claim 140, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 90% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO: 3 or a xylanase active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity comprising a polypeptide having at least 90% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.

142. The composition of claim 141, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 92% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity comprising a polypeptide having at least 92% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof.

143. The composition of claim 142, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity comprising a polypeptide having at least 95% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof.

144. The composition of claim 143, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 98% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity comprising a polypeptide having at least 98% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof.

145. The composition of claim 144, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NO: 2 or a xylanase- active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof.

146. The composition of claim 145, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide according to SEQ ID NO: 2 or a xylanase-active fragment thereof or SEQ ID NO: 3 or a xylanase-active fragment thereof, and wherein the beta-glucanase is an enzyme having beta-glucanase activity comprising a polypeptide according to SEQ ID NO: 8 or a beta-glucanase-active fragment thereof.

147. The composition of claim 146, wherein the xylanase is an enzyme having xylanase activity comprising a polypeptide according to SEQ ID NO: 2 or SEQ ID NO: 3, and wherein the beta-glucanase is an enzyme having beta-glucanase activity comprising a polypeptide according to SEQ ID NO:

8.

148. A method for producing a low viscosity mash, the method comprising the steps of: (a) preparing a mash from a cereal comminution comprising wheat kernels in the presence of a composition as claimed in any one of claims 137 to 147; (b) optionally filtering the mash to obtain wort.

149. The method of claim 148, wherein the wheat kernels are malted.

150. The method of claim 148, wherein the wheat kernels are un-malted.

151. The method of any one of claims 148 to 150, wherein the wheat kernels are milled.

152. The method of any one of claims 148 to 151, wherein the cereal comminution is 5, 10, 20, 25, 30, 35, 40, 45, 50, 51, 60, 70, 80, 85, 90, 95, 99, or 100 w / w% wheat.

153. The method of any one of claims 148 to 152, wherein the cereal comminution further comprises malted or un-malted barley, corn, rye, rice, cassava, oat flakes, or sorghum.

154. The method of claim 153, wherein the cereal comminution further comprises malted barley.

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