Novel methods and compositions for improving mash filterability and yield in brewing

Abstract

The present invention relates to improved techniques for brewing. More particularly, the present invention relates to compositions and methods for improving mash filterability and yield.

Description

[0001] TITLE

[0002] NOVEL METHODS AND COMPOSITIONS FOR IMPROVING MASH FILTERABILITY AND YIELD IN BREWING

[0003] TECHNICAL FIELD

[0004] The present invention relates to brewing. More particularly, the present invention relates to novel combinations of enzymes for improving mash filterability and yield.

[0005] REFERENCE TO A SEQUENCE LISTING

[0006] The contents of the electronic submission of the text file Sequence Listing, 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.

[0007] BACKGROUND OF THE INVENTION

[0008] In industrial beer brewing, the overall speed of the brewing process is of paramount importance. Parameters such as variations in raw material availability and quality, high gravity brewing, and climactic conditions and changes can impact the brewing process significantly.

[0009] During the mashing step, the starch present in malt and adjuncts must be gelatinized to achieve high extract yield and to convert carbohydrate polymers to fermentable sugar types. Other components from the raw materials get solubilized as well, e.g., high molecular weight (HMW) B-glucan and HMW arabinoxylan. The B-glucan and arabinoxylan are solubilized during the entire mashing process and until mashing-off. B-glucan and arabinoxylan are complex carbohydrates and cause the mash to be highly viscous, rendering subsequent mash separation and beer filtrations steps difficult. In turn, yields of fermentable sugars and, hence, ethanol is lowered.

[0010] It has been observed that the gelatinization temperature of the starch in barley and barley malt has increased in recent years, perhaps due to environmental factors. This requires adjustment of the mashing regime, including the use of higher temperatures. Furthermore, brewers today must often use adjunct and lower quality malt as sources of carbohydrates. Adjuncts include corn, rice, wheat, rye, cassava, and other non-traditional sources of fermentable sugars. Grist having adjunct frequently requires higher temperatures for mashing. It has been observed that prior art enzymes are inactivated at higher mashing temperatures. In addition, some adjuncts arc observed to have inhibitors that substantially reduce the activity of prior art enzymes.

[0011] There is a continuing need for methods and compositions for improving mash filterability and yield for the use of malt and adjunct of varying quality and composition.

[0012] SUMMARY OF THE INVENTION

[0013] In accordance with an aspect of the present invention, a method is presented for producing a rye whiskey having the steps of: (a) providing a grist having at least 51 w / w % of rye grain; (b) adding water to the grist 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 inhibitor; (f) saccharifying the mash of step (e); (g) fermenting the mash of step (f) to produce a fermentate containing ethanol; (h) distilling the fermentate of step (g) to provide the rye whiskey. Optionally, the xylanase has less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% inhibition by Rye XIP inhibitor. Optionally, the xylanase has less than 20% inhibition by Rye XIP inhibitor.

[0014] Optionally, the xylanase is an enzyme having xylanase activity with 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 with 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 with 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 with 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 with 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 with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID N0:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanasc active fragment thereof. Optionally, the xylanase is an enzyme having xylanase activity with 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 with 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 with 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 with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.

[0015] Optionally, the rye grain is malted. In other preferred embodiments, the rye grain is unmalted. Optionally, the rye grain is milled. Preferably, the grist is 60, 70, 80, 85, 90, 95, 99 or 100 w / w % rye. Optionally, the grist further comprises malted or unmalted barley, wheat, corn, rye, rice, cassava, oatmeal, or sorghum. More preferably, the grist further comprises malted barley.

[0016] Optionally, additional amounts of the rye uninhibited xylanase are added during any of steps (b), (c) and / or (d). Optionally, the method also includes the further step of adding an alphaamylase and / or a beta-glucanase at 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with a polypeptide amino acid sequence according to SEQ ID NO:8.

[0017] In the instantly disclosed method, optionally a glucoamylase is added at step (f) and / or a protease is added at step (g).

[0018] Optionally, the method further includes the step of adding an alpha-L- arabinofuranosidase at any of steps (b), (c), (d) and / or (e). Optionally, the alpha- L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity with 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. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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-arabinofuranosidasc active fragment thereof. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity with 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. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity with 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.

[0019] The disclosed method also optionally includes the further step of adding a feruloyl esterase at any of steps (b), (c), (d) and / or (e). Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence according to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

[0020] In another aspect of the present invention, a method is presented for preparing a low viscosity mash having the steps of: (a) preparing a mash from a grist having rye cereal in the presence of a xylanase having reduced sensitivity to rye XIP inhibitor; (b) optionally filtering the mash to obtain a wort. Optionally, the xylanase has less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% inhibition by Rye XIP inhibitor. Optionally, the xylanase has less than 20% inhibition by Rye XIP inhibitor.

[0021] Optionally, the xylanase is an enzyme having xylanase activity with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.

[0022] Optionally, the rye grain is malted. Optionally, the rye grain is unmalted. Optionally, the rye grain is milled. Optionally, the grist is 60, 70, 80, 85, 90, 95, 99 or 100 w / w % rye. In other preferred embodiments, the grist further comprises malted or unmalted barley, wheat, corn, rye, rice, cassava, oatmeal, or sorghum. Optionally, the grist further comprises malted barley.

[0023] Optionally, the method also includes the further step of adding an alpha-amylase and / or a beta-glucanase to the mash. Optionally, a beta-glucanase is added. Optionally, the beta- glucanase is an enzyme having beta-glucanase activity with 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 with 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 with 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 with 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 with 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 with 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 with 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 bcta-glucanasc is an enzyme having bcta-glucanasc activity with 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 with 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 with a polypeptide amino acid sequence according to SEQ ID NO:8.

[0024] Optionally, the method further includes the step of adding an alpha-L- arabinofuranosidase to the mash. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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. Optionally, the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity with 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. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidasc 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 with 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. Optionally, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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.

[0025] The disclosed method also optionally includes the further step of adding a feruloyl esterase to the mash. Optionally, the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence having 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 with a polypeptide amino acid sequence according to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

[0026] In another aspect of the present invention, a method is presented for identifying a xylanase that has reduced sensitivity to a cereal XIP inhibitor having the step of assaying the activity of the xylanase in the presence and absence of the cereal XIP inhibitor and determining the percent inhibition by the cereal XIP inhibitor. Optionally, the cereal is barley, wheat, corn, rye, rice, cassava, oatmeal, or sorghum. Optionally, the cereal is rye or wheat. Optionally, the cereal is rye. Optionally, the cereal is wheat.

[0027] In another aspect of the present invention, a xylanase is presented having reduced sensitivity to rye XIP inhibitor identified by the method above. Optionally, the xylanase has less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% inhibition by rye XIP inhibitor. Optionally, the xylanase has less than 20% inhibition by rye XIP inhibitor.

[0028] Optionally, the xylanase is an enzyme having xylanase activity with 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 with 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 with 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 with 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 with 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 with 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 with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID N0:2 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 with 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 with 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 with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.

[0029] In another aspect of the present invention, a xylanase is presented having reduced sensitivity to wheat XIP inhibitor identified by the method of claim 110. Optionally, the xylanase has less than 90, 80, 70, 60, 55, 50, 40, 30, 20, 10, 5 or 1% inhibition by wheat XIP inhibitor. Optionally, the xylanase has less than 55% inhibition by wheat XIP inhibitor. Optionally, the xylanase is an enzyme having xylanase activity with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.

[0030] In another aspect of the present invention, a composition is presented for reducing wort viscosity in wheat adjunct brewing having a xylanase having reduced sensitivity to wheat XIP inhibitor and a beta-glue anase. Optionally, the xylanase is an enzyme having xylanase activity with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 bcta-glucanasc is an enzyme having bcta-glucanasc activity with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with a polypeptide amino acid sequence according to SEQ ID NO:8.

[0031] In another aspect of the present invention, a method is presented for preparing a low viscosity mash having the steps of: (a) preparing a mash from a grist having wheat grain in the presence of a composition having a xylanase having reduced sensitivity to wheat XIP inhibitor and a beta-glucanase as described above; (b) optionally filtering the mash to obtain a wort. Optionally, the wheat grain is malted. Optionally, the wheat grain is unmalted. Optionally, the wheat grain is milled. Optionally, the grist 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 grist also has malted or unmalted barley, com, rye, rice, cassava, oatmeal, or sorghum. Optionally, the grist also has malted barley.

[0032] In another aspect of the present invention, a composition is presented having a xylanase which has a polypeptide sequence with at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof and a glucanase. Optionally, the xylanase polypeptide has at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase polypeptide has at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase polypeptide has at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase polypeptide has at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase polypeptide has at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase polypeptide has at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase polypeptide is a sequence according to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase polypeptide has a sequence according to SEQ ID NO:2.

[0033] Optionally, the glucanase is a polypeptide having at least 80% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase polypeptide has at least 85% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase polypeptide has at least 90% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase polypeptide has at least 92% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase polypeptide has at least 95% sequence identity to SEQ ID NO: 8 or a glucanase active fragment thereof. Optionally, the glucanase polypeptide has at least 98% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase polypeptide has at least 99% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase polypeptide is a sequence according to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase polypeptide is a sequence according to SEQ ID NO:8. Where the glucanase polypeptide has at least 95% sequence identity to SEQ ID NO:2, the xylanasc polypeptide optionally has at least 98, 99 or 100% sequence identity to SEQ ID NO:2.

[0034] Optionally, the composition is a liquid. In the liquid composition, the xylanase is optionally present in an amount of 1,000 to 50,000 NGXU / g and the glucanase is preferably present in an amount of 1,000 to 50,000 BBXU / g. Optionally, the xylanase is present in an amount of 5,000 to 30,000 NGXU / g and the glucanase is present in an amount of 5,000 to 30,000 BBXU / g. Optionally, the xylanase is present in an amount of 10,000 to 25,000 NGXU / g and the glucanase is present in an amount of 15,000 to 30,000 BBXU / g. Optionally, the xylanase is present in an amount of 15,000 to 20,000 NGXU / g and the glucanase is present in an amount of 20,000 to 28,000 BBXU / g.

[0035] In another aspect of the present invention, a method is presented of altering filterability of a starch containing material, the method having the step of treating the starch containing material with a composition as described above.

[0036] In another aspect of the present invention, a method is presented of reducing pressure built up during lautering in a brewing application, the method having the step of treating a brewing mash having a starch containing material with a composition as described above.

[0037] In another aspect of the present invention, a method is presented for the production of a food, feed, or beverage product, such as an alcoholic or non-alcoholic beverage, such as a cereal- or malt-based beverage like beer or whiskey, the method having the step of treating a starch containing material with a composition as described above.

[0038] In another aspect of the present invention, a method is presented for the production of a brewing mash, the method having the step of treating a starch comprising material with a composition as described above.

[0039] Optionally the starch comprising material comprises a grist. Preferably, the grist has at least 50, 60, 70, 80 or 90 % malt. Optionally, the grist further also contains wheat, corn or rye.

[0040] In the above methods, Optionally, 0.01 to 1 grams of the composition is added per Kg of grist. Optionally, 0.02 to 0.1 grams of the composition is added per Kg of grist.

[0041] In another aspect of the present invention, a method is for preparing a low viscosity mash having the steps of: (a) preparing a mash from a grist in the presence of a xylanase having reduced sensitivity to rye XIP inhibitor and a beta-glucanase which has increased activity in the presence of at least 10% rye compared to a sample not having rye; and (b) optionally filtering the mash to obtain a wort.

[0042] Optionally, the xylanase is a polypeptide having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase is a polypeptide having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase is a polypeptide having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase is a polypeptide having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase is a polypeptide having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase is a polypeptide having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase is a polypeptide having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase is a polypeptide according to SEQ ID NO:2 or a xylanase active fragment thereof. Optionally, the xylanase is a polypeptide according to SEQ ID NO:2.

[0043] Optionally, the glucanase is a polypeptide having at least 80% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase is a polypeptide having at least 85% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase is a polypeptide having at least 90% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase is a polypeptide having at least 92% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase is a polypeptide having at least 95% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase is a polypeptide having at least 98% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase is a polypeptide having at least 99% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase is a polypeptide according to SEQ ID NO:8 or a glucanase active fragment thereof. Optionally, the glucanase is a polypeptide according to SEQ ID NO:8.

[0044] Optionally, the grist has at least 50, 60, 70, 80 or 90% malt. Optionally, the grist also has wheat, com or rye. Optionally, the grist has rye. Optionally, the grist has at least 10% rye. BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

[0045] SEQ ID NO:1 shows the protein sequence of the FveXyn4_l precursor protein.

[0046] SEQ ID NO:2 shows the protein sequence of FveXyn4_l mature protein.

[0047] SEQ ID NO:3 shows the protein sequence of FveXyn4_l predicted protein.

[0048] SEQ ID NO:4 shows the amino acid sequence of PfunXyn GH11 xylanase (E.C. 3.2.1.8) precursor protein.

[0049] SEQ ID NO:5 shows the amino acid sequence of PfunXyn GH11 xylanase (E.C. 3.2.1.8) mature protein.

[0050] SEQ ID NO:6 shows the amino acid sequence of TemXyn GH10 xylanase (E.C. 3.2.1.8) precursor protein.

[0051] SEQ ID NOT shows the amino acid sequence of TemXyn GH10 xylanase (E.C. 3.2.1.8) mature protein.

[0052] SEQ ID NO:8 shows the amino acid sequence of BsbBglu mature protein (E.C. 3.2.1.6) beta-glucanase from Bacillus subtilis.

[0053] SEQ ID NO:9 shows the amino acid sequence of AkaXyl mature protein (E.C. 3.2.1.8.) xylanase variant from Aspergillus kawachii.

[0054] SEQ ID NOTO shows the amino acid sequence of AniAfur precursor protein alpha-L- Arabinofurano sidase .

[0055] SEQ ID NO: 11 shows the amino acid sequence of BaAfur precursor protein alpha-L- arabinofurano sidase .

[0056] SEQ ID NO: 12 shows the amino acid sequence of CtFe precursor protein feruloyl esterase.

[0057] BRIEF DESCRIPTION OF THE FIGURES

[0058] FIG. 1 depicts turbidity quantified by Anton Paar as haze by 90° scattering in EBC units (small particles) on mash supernatants. Haze scattering is shown for trials 3 and 6, 4 and 7, and 5 and 8 respectively with either PfunXyn or FvXyn4_l xylanase as indicated by legends (in combinations with a-L-arabinofuranose and Feruloyl esterase).

[0059] FIG. 2 depicts turbidity quantified by Anton Paar as haze by 25° scattering in EBC units (larger particles) on mash supernatants. Haze scattering is shown for trials 3 and 6, 4 and 7, and 5 and 8 respectively with either PfunXyn or FvXyn4_l xylanase as indicated by legends (in combinations with a-L-arabinofuranosc and Fcruloyl esterase).

[0060] FIG. 3 depicts the average wort volume collected after 30 minutes filtration for each xylanase over following dose range: TemXyn was dosed 0.27 to 0.69 U per g Grist and FveXyn4_l dosed 9.0 to 27.2 pg FveXyn4_l protein per g Grist.

[0061] FIG. 4 depicts the average wort Viscosity at 12°P (mPa.s) after filtration for each xylanase using following dosages: TemXyn was dosed 0.27 to 0.69 U per g Grist and FveXyn4_l dosed 9.0 to 27.2 pg FveXyn4_l protein per g Grist.

[0062] FIG. 5 depicts the average Original Extract (°P) of wort after filtration for each xylanase using following dosages: TemXyn was dosed 0.27 to 0.69 U per g Grist and FveXyn4_l dosed 9.0 to 27.2 pg FveXyn4_l protein per g Grist.

[0063] FIG. 6 depicts the effect of increasing xylanase (Viscoferm, PfunXyn and FveXyn4_l ) Dose Rate (g / kg grist) on Ethanol Production in rye-based SSF fermentation.

[0064] FIG. 7 depicts thermostability of FveXyn4_l, AkaXyl_var, Shearzyme P105 and Ultraflo Max in Malt wort pH 5.6 with 10 minutes incubation time. The residual xylanase activity is shown as function of temperature of wort.

[0065] FIG. 8 depicts residual xylanase activity through 40%:60% barley:malt mashing programs using a water-to-grist ratio of 3:1 of FveXyn4_l, AkaXyl_var, Shearzyme P105 and Ultrflo Max. Low (A), standard (B) and high (C) temperature mashing diagrams are shown with residual xylanase.

[0066] FIG. 9 depicts residual beta-glucanase activity (Beta-glucazyme, megazyme) of BsbBglu + FveXyn4_l, Ultraflo Max and Filtrase Fast incubated 24 hours at 5°C with 10% cereal extracts (Rye, Wheat, Com, and Malt).

[0067] FIG. 10 depicts wort viscosity (mPa*s) (A), HWM b-glucan (mg / L) (B) and HWM pentosan (mg / L) (C) for BsbBglu + FveXyn4_l, Ultraflo Max and BsbBglu + AkaXyl_var in wort produced by 40%:60% barley:malt high temperature mashing. DETAILED DESCRIPTION

[0068] Definitions

[0069] An “active fragment” of an enzyme is a polypeptide where the enzyme has been deleted either at the C-terminus, the N-terminus and / or internally but still retains some or all of its original activity. An active fragment includes the mature form of an enzyme.

[0070] In addition to the specific amino acid sequences and polynucleotides mentioned herein, the present invention encompasses variants, homologues, derivatives and fragments thereof. The term "variant" is used to mean a nucleotide sequence or amino acid sequence which differs from a wild-type sequence.

[0071] For example, a variant may include substitutions, insertions, deletions, truncations, transversions and / or inversions at one or more position(s) relative to a wild-type sequence. Variants can be made using methods known in the art for example site scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis and directed-evolution as well as using recombinant methods well known in the art. Polynucleotide sequences encoding variant amino acid sequences may readily be synthesized using methods known in the art.

[0072] In some aspects, the variant is a naturally occurring nucleotide sequence or amino acid sequence which differs from a wild-type sequence. For example, the variant may be a natural genetic variant.

[0073] In some aspects, the variant is an engineered variant. For example, the variant may be engineered by recombinant methods.

[0074] The protein sequences of the instant invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions may be made, for example according to the Table below.

[0075] Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other as set forth in Table 1.

[0076] Table 1

[0077] The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e., like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e., from one class of residue to another or alternatively involving the inclusion of unnatural 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), pyriylalanine, thienylalanine, naphthylalaninc and phcnylglycinc.

[0078] Replacements may also be made by synthetic amino acids (e.g. unnatural amino acids) include; alpha* and alpha-disubstituted* 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*, B-alanine*, L-a-amino butyric acid*, L-g- amino butyric acid*, L-a-amino isobutyric acid*, L-e-amino caproic acid#, 7-amino heptanoic acid*, L- methionine sulfone**, L-norleucine*, L-norv aline*, p-nitro-L-phenylalanine*, L- hydroxyproline*, L- thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl- Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (l,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid* and L-Phe (4- benzyl)*. The notation * has been utilized for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas # has been utilized to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.

[0079] Variant amino acid sequences may include suitable spacer groups that may 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. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, “the peptoid form” is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue’s nitrogen atom rather than the a-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ el al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

[0080] Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridizing to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries or genomic DNA libraries made from other animal species and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.

[0081] Variants and strain / species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. 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.

[0082] The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences. Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterized sequences. This may be useful where for example silent codon sequence changes are required to optimize codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

[0083] The present invention employs, unless otherwise indicated, conventional techniques of biochemistry, molecular biology, microbiology and recombinant DNA, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); 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 these general texts is herein incorporated by reference.

[0084] As used herein, “percent (%) sequence identity” means that a particular sequence has at least a certain percentage of amino acid residues identical to those 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. Default parameters for the CLUSTAL W algorithm are:

[0085] Gap opening penalty: 10.0

[0086] Gap extension penalty: 0.05

[0087] Protein weight matrix: BLOSUM series

[0088] DNA weight matrix: IUB

[0089] Delay divergent sequences %: 40

[0090] Gap separation distance: 8

[0091] DNA transitions weight: 0.50

[0092] List hydrophilic residues: GPSNDQEKR Use negative matrix: OFF

[0093] Toggle Residue specific penalties: ON

[0094] Toggle hydrophilic penalties: ON

[0095] Toggle end gap separation penalty: OFF

[0096] Deletions are counted as non-identical residues, compared to a reference sequence. Deletions occurring at either terminus are included. For example, a variant with five amino acid deletions of the C-terminus of the mature 617 residue polypeptide would have a percent sequence identity of 99% (612 / 617 identical residues x 100, rounded to the nearest whole number) relative to the mature polypeptide. Such a variant would be encompassed by a variant having “at least 99% sequence identity” to a mature polypeptide.

[0097] In accordance with the instant invention, proteins, including enzymes, of the present invention exist in multiple forms. Proteins of the instant invention may be clipped or trimmed (i.e., removing amino acids) from the N-terminus and / or the C-terminus, resulting in a shorter protein. Proteins of the instant invention can also have internal deletions. Shorter proteins as described herein can have higher activity or lower activity than longer counterparts. Without being bound by theory, as used herein the term precursor protein is a protein, including an enzyme, which has an N-terminal signal peptide that targets the protein for secretion. A precursor protein is sometimes referred to herein as “full length” or “full length protein”. The N- terminal signal peptide is cleaved off in the endoplasmic reticulum to yield a mature protein.

[0098] Xylanases are classified in EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.136 and EC 3.2.1.156.; their activity may be measured e.g., as described in the examples. Suitable xylanases to be used in combination with an enzyme exhibiting endo-l,3(4)-P-glucanase activity according to the invention includes any xylanase classified in EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.136 and EC 3.2.1.156, such as anyone disclosed in WO 2010072226, WO 2010072225, WO 2010072224, WO 2005059084, W02007056321, W02008023060A, WO9421785, W02006114095, W02006066582, US 2008233175, and W010059424.

[0099] Endo-P-1, 4-xylanases, also referred herein as xylanases, is the name given to a class of enzymes which degrade the linear polysaccharide beta- 1,4- xylan into xylose, and 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. The enzyme causes endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans. The xylanases of the present invention are superior for solubilization of rye fibers, both un-cxtractablc arabinoxylans (WU-AX) and the water extractable arabinoxylans (WE- AX). In addition, xylanases of the instant invention have excellent product properties relevant for e.g., mashing applications and high thermostability.

[0100] The terms “Family 10 xylanase”, “Glycoside hydrolase (GH) family 10”, or simply “GH

[0101] 10 xylanase” comprises enzymes with a number of known activities, such as xylanase (EC:3.2.1.8); endo-l,3-beta-xylanase (ECG.2.1.32); cellobiohydrolase (EC:3.2.1.91). These enzymes were formerly known as cellulase family F.

[0102] The terms “family 11 xylanase”, “Glycoside hydrolase (GH) family 11” or simply “GH

[0103] 11 xylanase” as used herein refers to an endo- 1 ,4-beta xylanase classified as EC 3.2.1.8, which causes endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans and which is classified as a family 11 xylanase according to B. Henrissat, A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280 (1991), pp. 309-316.

[0104] A xylanase variant described in the present invention is superior for solubilization of Rye fiber, both un-extractable arabinoxylans (WU-AX) and the water extractable arabinoxylans (WE- AX). This property of the xylanase when used during mashing of Rye-based substrate provides enhanced filterability and / or improved extract yield after filtration.

[0105] All references cited in the present specification are hereby incorporated by reference in their entirety. In particular’, the teachings of all references herein specifically referred to are incorporated by reference.

[0106] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0107] By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0108] Other definitions are set forth below.

[0109] Production of enzymes

[0110] The enzymes of the present invention can be produced in host cells, for example, by secretion or intracellular expression. A cultured cell material (e.g., a whole-cell broth) having an enzyme can be obtained following secretion of the enzyme into the cell medium. Optionally, the enzyme can be isolated from the host cells, or even isolated from the cell broth, depending on the desired purity of the final enzyme. Suitable host cells include bacterial, fungal (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, e.g., Bacillus subtilis or B. licheniformis. as well as Streptomyces, E. coli.

[0111] Vectors

[0112] A DNA construct comprising a nucleic acid encoding an enzyme can be constructed to be expressed in a host cell. Because of the well-known degeneracy in the genetic code, variant polynucleotides that encode an identical amino acid sequence can be designed and made with routine skill. It is also well-known in the art to optimize codon use for a particular host cell. Nucleic acids encoding xylanase can be incorporated into a vector. Vectors can be transferred to a host cell using well-known transformation techniques, such as those disclosed below.

[0113] The vector may be any vector that can be transformed into and replicated within a host cell. For example, a vector comprising a nucleic acid encoding an enzyme can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector. The vector also may be transformed into an expression host, so that the encoding nucleic acids can be expressed as a functional xylanase. Host cells that serve as expression hosts can include filamentous fungi, for example. The Fungal Genetics Stock Center (FGSC) Catalogue of Strains lists suitable vectors for expression in fungal host cells. See FGSC, Catalogue of Strains, University of Missouri, at www.fgsc.net (last modified January 17, 2007). A representative vector is pJG153, a promoterless Cre expression vector that can be replicated in a bacterial host. See Harrison et al. (June 2011) Applied Environ. Microbiol. 77: 3916-22. pJG153can be modified with routine skill to comprise and express a nucleic acid encoding a xylanase.

[0114] A nucleic acid encoding an enzyme can be operably linked to a suitable promoter, which allows transcription in the host cell. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Exemplary promoters for directing the transcription of the DNA sequence encoding a xylanase, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA or celA promoters, the promoters of the Bacillus licheniformis a-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens a-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. 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 a-amylase, A. niger acid stable a-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase. When a gene encoding an enzyme is expressed in a bacterial species such as E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. Examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris A0X1 or AOX2 promoters, cbhl is an endogenous, inducible promoter from Trichoderma reesei. See Liu el al. (2008) “Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbhl) promoter optimization,” Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.

[0115] The coding sequence can be operably linked to a signal sequence. The DNA encoding the signal sequence may be the DNA sequence naturally associated with the enzyme gene to be expressed or from a different Genus or species. A signal sequence and a promoter sequence comprising a DNA construct or vector can be introduced into a fungal host cell and can be derived from the same source. For example, the signal sequence is the cbhl signal sequence that is operably linked to a cbhl promoter.

[0116] An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding a variant xylanase. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

[0117] The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBUO, pE194, pAMBl, and pIJ702.

[0118] The vector may also comprise a selectable marker, e.g., a gene the product of which 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 such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and xx.sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, such as known in the art. See e.g., International PCT Application WO 91 / 17243.

[0119] Intracellular expression may be advantageous in some respects, e.g., when using certain bacteria or fungi as host cells to produce large amounts of xylanase for subsequent enrichment or purification. Extracellular secretion of xylanase into the culture medium can also be used to make a cultured cell material comprising the isolated xylanase.

[0120] The expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes. The expression vector normally comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the enzyme to a host cell organelle such as a peroxisome, or to a particular host cell compartment. Such a targeting sequence includes but is not limited to the sequence, SKL. For expression under the direction of control sequences, the nucleic acid sequence of the xylanase is operably linked to the control sequences in proper manner with respect to expression.

[0121] The procedures used to ligate the DNA construct encoding an 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, e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nded., Cold Spring Harbor, 1989, and 3rded., 2001).

[0122] Transformation and Culture of Host Cells

[0123] An isolated cell, either comprising a DNA construct or an expression vector, is advantageously used as a host cell in the recombinant production of an enzyme according to the instant invention. The cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

[0124] Examples of suitable bacterial host organisms are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (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 a Gram negative bacterial species belonging to Enterobacteriaceae including E. coli, or to Pseudomonadaceae can be selected as the host organism.

[0125] A suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces, including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces such as, for example, 5. pombe species. A strain of the methylotrophic yeast species, Pichia pastoris, can be used as the host organism. Alternatively, the host organism can be a Hansenula species. Suitable host organisms among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively, strains of a Fusarium species, e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. Other suitable strains include Thermomyces and Mucor species. In addition, Trichoderma sp. can be used as a host. A suitable procedure for transformation of Aspergillus host cells includes, for example, that described in EP 238023. An enzyme expressed by a fungal host cell can be glycosylated, i.e., will comprise a glycosyl moiety. The glycosylation pattern can be the same or different as present in the wild-type xylanase. The type and / or degree of glycosylation may impart changes in enzymatic and / or biochemical properties.

[0126] It may be advantageous to delete genes from expression hosts, where the gene deficiency can be cured by the transformed expression vector. Known methods may be used to obtain a fungal host cell having one or more inactivated genes. Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means that renders a gene nonfunctional for its intended purpose, such that the gene is prevented from expression of a functional protein. Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned can be deleted, for example, cbhl, cbh2. egll, and egl2 genes. Gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.

[0127] Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, e.g., 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. The expression of heterologous protein in Trichoderma is described, for example, in U.S. Patent No. 6,022,725. Reference is also made to Cao et al. (2000) Science 9:991-1001 for transformation of Aspergillus strains. Genetically stable transformants can be constructed with vector systems whereby the nucleic acid encoding an enzyme is stably integrated into a host cell chromosome. Transformants are then selected and purified by known techniques.

[0128] Expression

[0129] A method of producing an enzyme of the instant invention may comprise cultivating a host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and / or culture medium.

[0130] The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the enzyme. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).

[0131] An enzyme secreted from the host cells can be used in a whole broth preparation. In the present methods, the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the ail resulting in the expression of a xylanase. Fermentation may, therefore, be understood as comprising shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed-batch, or solid- state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the xylanase to be expressed or isolated. The term “spent whole fermentation broth” is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is understood that the term “spent whole fermentation broth” also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.

[0132] An enzyme secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

[0133] Host cells may be cultured under suitable conditions that allow expression of a xylanase. Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or Sophorose. Polypeptides can also be produced recombinantly in an in vitro cell-free system, such as the TNT™ (Promega) rabbit reticulocyte system.

[0134] Methods for Enriching and Purifying enzymes

[0135] Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used in order to prepare an enzyme polypeptide-containing solution.

[0136] After fermentation, a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain an enzyme solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultrafiltration, extraction, or chromatography, or the like, are generally used.

[0137] It is desirable to concentrate an enzyme polypeptide-containing solution in order to optimize recovery. Use of unconcentrated solutions requires increased incubation time in order to collect the enriched or purified enzyme precipitate.

[0138] The enzyme containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme containing solution may 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.

[0139] DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0140] In accordance with an 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 employed in distilling and brewing to overcome longstanding problems. U.S. law requires that spirits sold as rye whiskey must be produced from malts composed of at least 51% rye grains. It is known to those of skill in the art that such malts are exceedingly difficult to work with on an industrial scale.

[0141] The present invention relates to a rye mashing and an optional filtration step in a process for the production of an alcoholic beverage, such as beer or distilling applications such as whiskey, and to a composition useful in the mashing and optional filtration step in such a process.

[0142] The use of xylanases to improve filterability of malt-based mashes is known. However, in accordance with the present invention, it has been discovered that specific proteinaceous inhibitors of xylanases are present in non-malt adjuncts such as rye and wheat. Methods are provided in accordance with the instant invention for screening xylanases to find those that are less inhibited by wheat and rye factors.

[0143] The present invention discloses a xylanase with very low rye and wheat XIP inhibition that show excellent properties in decreasing the viscosity or rye and wheat based mashes. In beer brewing, it is common to filter the mash to prepare a liquid wort for subsequent operations. Here, the lower viscosity provides greater filterability. In distilling and in some beer brewing, the mash goes to fermentation without filtration. Here, lower mash viscosity results in less gumming of fermentation and distilling equipment and a higher yield of ethanol.

[0144] In accordance with an aspect of the present invention, a method is presented for producing a rye whiskey having the steps of: (a) providing a grist comprising 51 w / w % of rye grain; (b) adding water to the grist 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 inhibitor; (f) saccharifying the mash of step (e); (g) fermenting the mash of step (f) to produce a fermentate containing ethanol; (h) distilling the fcrmcntatc of step (g) to provide the rye whiskey. Preferably, the xylanasc has less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% inhibition by Rye XIP inhibitor. More preferably, the xylanase has less than 20% inhibition by Rye XIP inhibitor.

[0145] Preferably, the xylanase is an enzyme having xylanase activity with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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. Still more preferably, the xylanase is an enzyme having xylanase activity with 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 with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3. Preferably, the rye grain is malted. In other preferred embodiments, the rye grain is unmaltcd. Preferably, the rye grain is milled. Preferably, the grist is 60, 70, 80, 85, 90, 95, 99 or 100 w / w % rye. In other preferred embodiments, the grist further comprises malted or unmalted barley, wheat, com, rye, rice, cassava, oatmeal or sorghum. More preferably, the grist further comprises malted barley.

[0146] Preferably, additional amounts of the rye uninhibited xylanase are added during any of steps (b), (c) and / or (d). Preferably, the method also includes the further step of adding an alpha-amylase and / or a beta-glucanase at 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with a polypeptide amino acid sequence according to SEQ ID NO:8.

[0147] In the instantly disclosed method, preferably, a glucoamylase is added at step (f) and / or a protease is added at step (g).

[0148] Preferably, the method further includes the step of adding an alpha-L- arabinofuranosidase at any of steps (b), (c), (d) and / or (e). Preferably, the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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 with 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 with 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 with 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 with 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 with 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 with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10 or an alpha-L- arabinofurano sidasc 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 with 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 embodiments, the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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.

[0149] The disclosed method also preferably includes the further step of adding a feruloyl esterase at any of steps (b), (c), (d) and / or (e). Preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with a polypeptide amino acid sequence according to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

[0150] In another aspect of the present invention, a method is presented for preparing a low viscosity mash having the steps of: (a) preparing a mash from a grist having rye cereal in the presence of a xylanase having reduced sensitivity to rye XIP inhibitor; (b) optionally filtering the mash to obtain a wort. Preferably, the xylanase has less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% inhibition by Rye XIP inhibitor. More preferably, the xylanase has less than 20% inhibition by Rye XIP inhibitor.

[0151] Preferably, the xylanase is an enzyme having xylanase activity with 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 with 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 with 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, an enzyme having xylanase activity with 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 with 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 with 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 with 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 with 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. Still more preferably, the xylanase is an enzyme having xylanase activity with 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 with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.

[0152] Preferably, the rye grain is malted. In other preferred embodiments, the rye grain is unmalted. Preferably, the rye grain is milled. Preferably, the grist is 60, 70, 80, 85, 90, 95, 99 or 100 w / w % rye. In other preferred embodiments, the grist further comprises malted or unmalted barley, wheat, com, rye, rice, cassava, oatmeal or sorghum. More preferably, the grist further comprises malted barley.

[0153] Preferably, the method also includes the further 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with a polypeptide amino acid sequence according to SEQ ID NO:8.

[0154] Preferably, the method further includes the step of adding an alpha-L- arabinofuranosidase to the mash. Preferably, the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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 with 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 with 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 with 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 with 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 with 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 with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10 or an alpha-L- arabinofurano sidasc 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 with 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 embodiments, the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with 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.

[0155] The disclosed method also preferably includes the further step of adding a feruloyl esterase to the mash. Preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with a polypeptide amino acid sequence according to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.

[0156] In another aspect of the present invention, a method is presented for identifying a xylanase that has reduced sensitivity to a cereal XIP inhibitor having the step of assaying the activity of the xylanase in the presence and absence of the cereal XIP inhibitor and determining the percent inhibition by the cereal XIP inhibitor. Preferably, the cereal is barley, wheat, corn, rye, rice, cassava, oatmeal, 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.

[0157] In another aspect of the present invention, a xylanase is presented having reduced sensitivity to rye XIP inhibitor identified by the method above. Preferably, the xylanase has less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% inhibition by rye XIP inhibitor. Preferably, the xylanase has less than 20% inhibition by rye XIP inhibitor.

[0158] Preferably, the xylanase is an enzyme having xylanase activity with 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 with 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 with 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, an enzyme having xylanase activity with 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 with 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 with 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 with 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 with 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. Still more preferably, the xylanase is an enzyme having xylanase activity with 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 with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.

[0159] In another aspect of the present invention, a xylanase is presented having reduced sensitivity to wheat XIP inhibitor identified by the method of claim 110. Preferably, the xylanase has less than 90, 80, 70, 60, 55, 50, 40, 30, 20, 10, 5 or 1% inhibition by wheat XIP inhibitor. More preferably, the xylanase has less than 55% inhibition by wheat XIP inhibitor.

[0160] Preferably, the xylanase is an enzyme having xylanase activity with 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 with 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 with 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, an enzyme having xylanase activity with 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 with 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 with 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 with 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 with 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. Still more preferably, the xylanase is an enzyme having xylanase activity with 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 with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.

[0161] In another aspect of the present invention, a composition is presented for reducing wort viscosity in wheat adjunct brewing having a xylanase having reduced sensitivity to wheat XIP inhibitor and a beta-glucanase. Preferably, the xylanase is an enzyme having xylanase activity with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with a polypeptide amino acid sequence according to SEQ ID NO:8 or a beta-glucanase active fragment thereof. In the most preferred embodiments, the xylanase is an enzyme having xylanase activity with 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 with a polypeptide amino acid sequence according to SEQ ID NO:8.

[0162] In another aspect of the present invention, a method is presented for preparing a low viscosity mash having the steps of: (a) preparing a mash from a grist having wheat grain in the presence of a composition having a xylanase having reduced sensitivity to wheat XTP inhibitor and a bcta-glucanasc as described above; (b) optionally filtering the mash to obtain a wort.

[0163] Preferably, the wheat grain is malted. In other preferred embodiments, the wheat grain is unmalted. Preferably, the wheat grain is milled. Preferably, the grist 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 grist also has malted or unmalted barley, corn, rye, rice, cassava, oatmeal, or sorghum. More preferably, the grist also has malted barley.

[0164] In another aspect of the present invention, a composition is presented having a xylanase which has a polypeptide sequence with at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof and a glucanase. Preferably, the xylanase polypeptide has at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. More preferably, the xylanase polypeptide has at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Still more preferably, the xylanase polypeptide has at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Yet more preferably, the xylanase polypeptide has at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. In yet more preferably embodiments, the xylanase polypeptide has at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Still more preferably, the xylanase polypeptide has at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Yet more preferably, the xylanase polypeptide is a sequence according to SEQ ID NO:2 or a xylanase active fragment thereof. In the most preferred embodiments, the xylanase polypeptide has a sequence according to SEQ ID NO:2.

[0165] Preferably, the glucanase is a polypeptide having at least 80% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. More preferably, the glucanase polypeptide has at least 85% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Still more preferably, the glucanase polypeptide has at least 90% sequence identity to SEQ ID NO: 8 or a glucanase active fragment thereof. In yet more preferred embodiments, the glucanase polypeptide has at least 92% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. More preferably, the glucanase polypeptide has at least 95% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Yet more preferably, the glucanase polypeptide has at least 98% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Still more preferably, the glucanase polypeptide has at least 99% sequence identity to SEQ ID NO: 8 or a glucanase active fragment thereof. In yet more preferred embodiments, the glucanase polypeptide is a sequence according to SEQ ID NO: 8 or a glucanase active fragment thereof. In the most preferred embodiments, the glucanase polypeptide is a sequence according to SEQ ID NO:8.

[0166] Where the glucanase polypeptide has at least 95% sequence identity to SEQ ID NO:2, the xylanase polypeptide preferably has at least 98, 99 or 100% sequence identity to SEQ ID NO:2.

[0167] Preferably, the composition is a liquid. In the liquid composition, the xylanase is preferably present in an amount of 1,000 to 50,000 NGXU / g and the glucanase is preferably present in an amount of 1,000 to 50,000 BBXU / g. More preferably, the xylanase is present in an amount of 5,000 to 30,000 NGXU / g and the glucanase is present in an amount of 5,000 to 30,000 BBXU / g. Still more preferably, the xylanase is present in an amount of 10,000 to 25,000 NGXU / g and the glucanase is present in an amount of 15,000 to 30,000 BBXU / g. In the most preferred embodiments, the xylanase is present in an amount of 15,000 to 20,000 NGXU / g and the glucanase is present in an amount of 20,000 to 28,000 BBXU / g.

[0168] In another aspect of the present invention, a method is presented of altering filterability of a starch containing material, the method having the step of treating the starch containing material with a composition as described above.

[0169] In another aspect of the present invention, a method is presented of reducing pressure built up during lautering in a brewing application, the method having the step of treating a brewing mash having a starch containing material with a composition as described above.

[0170] In another aspect of the present invention, a method is presented for the production of a food, feed, or beverage product, such as an alcoholic or non-alcoholic beverage, such as a cereal- or malt-based beverage like beer or whiskey, the method having the step of treating a starch containing material with a composition as described above.

[0171] In another aspect of the present invention, a method is presented for the production of a brewing mash, the method having the step of treating a starch comprising material with a composition as described above.

[0172] Preferably, the starch comprising material comprises a grist. Preferably, the grist has at least 50, 60, 70, 80 or 90 % malt. More preferably, the grist further also contains wheat, corn or rye. In the above methods, preferably 0.01 to 1 grams of the composition is added per Kg of grist. More preferably, 0.02 to 0.1 grams of the composition is added per Kg of grist.

[0173] In another aspect of the present invention, a method is for preparing a low viscosity mash having the steps of: (a) preparing a mash from a grist in the presence of a xylanase having reduced sensitivity to rye XIP inhibitor and a beta-glucanase which has increased activity in the presence of at least 10% rye compared to a sample not having rye; and (b) optionally filtering the mash to obtain a wort.

[0174] Preferably, the xylanase is a polypeptide having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. More preferably, the xylanase is a polypeptide having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Still more preferably, the xylanase is a polypeptide having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Yet more preferably, the xylanase is a polypeptide having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. In still more preferred embodiments, the xylanase is a polypeptide having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. More preferably, the xylanase is a polypeptide having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Still more preferably, the xylanase is a polypeptide having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Yet more preferably, the xylanase is a polypeptide according to SEQ ID NO:2 or a xylanase active fragment thereof. Most preferably, the xylanase is a polypeptide according to SEQ ID NO:2.

[0175] Preferably, the glucanase is a polypeptide having at least 80% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. More preferably, the glucanase is a polypeptide having at least 85% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Yet more preferably, the glucanase is a polypeptide having at least 90% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. In still more preferred embodiments, the glucanase is a polypeptide having at least 92% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. More preferably, the glucanase is a polypeptide having at least 95% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Still more preferably, the glucanase is a polypeptide having at least 98% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Yet more preferably, the glucanase is a polypeptide having at least 99% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. In still more preferred embodiments, the glucanase is a polypeptide according to SEQ ID NO:8 or a glucanasc active fragment thereof. Most preferably, the glucanase is a polypeptide according to SEQ ID NO:8.

[0176] Preferably, the grist has at least 50, 60, 70, 80 or 90% malt. More preferably, the grist also has wheat, corn or rye. More preferably, the grist has rye. Still more preferably, the grist has at least 10% rye.

[0177] EXAMPLES

[0178] The present disclosure is described in further detail in the following examples, which are not in any way intended to limit the scope of the disclosure as claimed. The attached figures are meant to be considered as integral parts of the specification and description of the disclosure. The following examples are offered to illustrate, but not to limit the claimed disclosure.

[0179] Example 1 - Enzymes

[0180] FveXyn4_l is a mature xylanase variant from Fusarium verticilliodes having the amino acid sequence shown in SEQ ID NO:2.

[0181] As an example of a very efficient solubilizing xylanase used in brewing, a preparation of the GH11 xylanase (E.C. 3.2.1.8) from Penicillium funiculosum was utilized (PfunXyn) having the mature amino acid sequence shown in SEQ ID NO:5. This preparation of PfunXyn (having an activity of 3150 CMC-DNS U / g) was provided from Dupont Nutrition Bioscience Aps (Denmark). Another example of an efficient solubilizing xylanase used in brewing, a preparation of the GH10 xylanase (E.C. 3.2.1.8) from Talaromyces (Geosmithia) emersonii was utilized (TemXyn) having the mature amino acid sequence shown in SEQ ID NO:7. This preparation of TemXyn (having an activity of 5525 units / g) was provided from Dupont Nutrition Bioscience Aps (Denmark). In addition, as an example of an enzyme complex hydrolyzing betaglucans and non-starch polysaccharides like arabinoxylans, containing a (E.C. 3.2.1.6) beta- glucanase from Bacillus subtilis (BsbBglu) having the mature amino acid sequence shown in SEQ ID NO:8 and a (E.C. 3.2.1.8) Xylanase variant from Aspergillus kawachii (AkaXyl_var) having the mature amino acid sequence shown in SEQ ID NO:9 was utilized. This preparation of BsbBglu + AkaXyl_var (having a xylanase activity of 13680 - 16720 XBU / g and having beta- glucanase activity of 9090 - 12500 BBU / g) was provided from Dupont Nutrition Bioscience Aps (Denmark). As an example of an enzyme complex having various cellulytic activities including bcta-glucosidasc (EC3.2.1.21) and xylanasc produced by Trichoderma reesei (Hypocrea jecorind) TrXyl was obtained from Dupont Nutrition Bioscience Aps (Denmark), beta- glucanase from Bacillus subtilis (BsdBglu) (beta-glucanase activity of 51000 U / g) was provided from Dupont Nutrition Bioscience Aps (Denmark).

[0182] As an example of a a-L-Arabinofuranosidase with potential hydrolysis of hydrolysis of terminal, non-reducing a-L-arabinofuranose from singly substituted xylose residues in arabinoxylan (a- 1,2 > a- 1,3), AniAfur, an a-L-Arabinofuranosidase from Aspergillus nidulans (89U / mg 500U) was sourced from Megazyme International, Ireland. AniAfur would have a pH optimum of 4.5 and temperature optimum of 40°C. As another example of an a-L- Arabinofuranosidase, BaAfur, an a-L-Arabinofuranosidase from Bifidobacterium adolescentis, (102U / mg 400U) was sourced from Megazyme International, Ireland. BaAfur would have a pH optimum of 6.0 and temperature optimum of 50°C. This enzyme would be expected to be highly specific hydrolysis of a-l,3-linked L-arabinofuranose residues from doubly substituted D- xylosyl or L-arabinosyl residues of arabinoxylans and branched arabinans, respectively.

[0183] As an example of a Feruloyl esterase, CtFe, a Feruloyl esterase from Clostridium thermocellum (0.5U / mg 100U) was sourced from Megazyme International, Ireland. CtFe would have a pH optimum of 6.0 and a temperature optimum of 60°C. CtFe is expected to catalyse the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar, which is usually arabinose in "natural" substrates.

[0184] Example 2 - Cloning of Fusarium verticillioides xylanase (FveXyn4)

[0185] Genomic DNA isolated from a strain of Fusarium verticillioides was used for amplifying a xylanase gene. The sequence of the cloned gene, called the FveXyn4 was recombinantly changed as described in WO2015 / 114108. The protein encoded by the FveXyn4_l gene is depicted in SEQ ID No. 1. The protein product of gene FveXyn4 belongs to glycosyl hydrolase family 10 (GH10) based on the PF AM search (http: / / pfam.sanger.ac.uk / ). At the N-terminus, FveXyn4 protein has a 25 amino acid signal peptide predicted by SignalP-NN (Emanuels son et al., Nature Protocols, 2:953-971, 2007). This indicates that FveXyn4 is a secreted glycosyl hydrolase. Example 3 - Expression of Fusarium verticillioides xylanase variant FveXyn4_l

[0186] The FvcXyn4_l gene was amplified from genomic DNA of Fusarium verticillioides variant 1 using the following primers: Primer 1 5'-caccATGAAGCTGTCTTCTTTCCTCTA-3', and Primer 2 5'-TTTTTAGCGGAGAGCGTTGACAACAGC-3'. The PCR product was cloned into pENTR / D-TOPO vector (invitrogen K2400) to generate the FveXyn4_l pEntry plasmid. The expression plasmid pZZH254 was obtained by Gateway cloning reaction between the FveXyn4_l pEntry plasmid and pTrex3gM expression vector (described in US 2011 / 0136197 Al) using Gateway® LR Clonase® II enzyme kit (Invitrogen 11791). The sequence of the FveXyn4 gene was confirmed by DNA sequencing. The plasmid pZZH254 was transformed into a quad deleted Trichoderma reesei strain (described in WO 05 / 001036) using biolistic method (Te'o VS et al., J Microbiol Methods, 51:393-9, 2002).

[0187] Following sequence confirmation, protoplasts of a quad deleted T. reesei strain (described in WO 05 / 001036) were transformed with the expression plasmid pTTT-Ate CAI using the PEG protoplast method (Penttila et al, Gene, 61:155-164, 1987). For protoplast preparation, spores were grown for about 10 hours at 24°C 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 CaC12x2H2O, 1 ml of 1000X T. reesei Trace elements solution (175 g / L Citric Acid anhydrous, 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). Germinating spores were harvested by centrifugation and treated with 30 mg / mL Vinoflow FCE (Novozymes, AG Switzerland) solution for from 7 hours to overnight at 30°C at 100 rpm to lyse the fungal cell walls. Protoplasts were washed in 0.1 M Tris HC1 buffer (pH 7) containing 0.6 M sorbitol and resuspended in 10 mM Tris HC1 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 107 protoplasts in a total volume of 200 pl 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 CaC12 solution. Transformants were selected on a medium containing acetamide as a sole source of nitrogen (acetamide 0.6 g / L; cesium chloride 1.68 g / L; glucose 20 g / L; potassium dihydrogen phosphate 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 (11) sulfate 1.6 mg / L; agar 20 g / L; pH 4.25). Transformed colonies (about 50-100) appeared in about 1 week. After growth on acetamide plates, the spores were collected and reselected on acetamide plates. After 5 days, the spores were collected using 10% glycerol, and 1 x 108 spores were inoculated in a 250 ml shake flask with 30 ml Glucosc / Sophorosc defined medium for protein expression. Protein expression was confirmed by SDS-PAGE. The spore suspension was subsequently grown in a 7 L fermentor in a defined medium containing 60% gluco se-sophorose feed. Glucose / Sophorose defined medium (per liter) consists of (NH4)2SO4 5 g, PIPPS buffer 33 g, Casamino Acids 9 g, KH2PO4 4.5 g, CaC12 (anhydrous) 1 g, MgSO4.7H2O 1 g, pH to 5.5 adjusted with 50% NaOH with Milli-Q H2O to bring to 966.5 mL. After sterilization, the following were added: 26 mL 60% Glucose / Sophrose, and 400X T. reeselTrace Metals 2.5 mL.

[0188] FveXyn4_l was purified from concentrated fermentation broth of a 7L fermentor 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 on a hydrophobic interaction chromatography column (Sepharose Phenyl FF, 26 / 10). The protein was eluted from the column using a linear gradient of equilibration / wash buffer to 20 mM sodium phosphate buffer pH 6.0. The fraction containing FveXyn4_l protein was 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. The purified protein was concentrated using a 3K Amicon Ultra- 15 device and the concentrated protein fraction was used in further studies.

[0189] Example 4 - Protein Determination Methods

[0190] Protein Determination by Stain Free Imager Criterion

[0191] Protein was quantified by SDS-PAGE gel and densitometry using Gel Doc™ EZ imaging system. Reagents used in the assay: Concentrated (2x) Laemmli Sample Buffer (Bio-Rad, Catalogue #161-0737); 26-well XT 4-12% Bis-Tris Gel (Bio-Rad, Catalogue #345-0125); protein markers “Precision Plus Protein Standards” (Bio-Rad, Catalogue #161- 0363); protein standard BSA (Thermo Scientific, Catalogue #23208) and SimplyBlue Safestain (Invitrogen, Catalogue #LC 6060. The assay was carried out as follow: In a 96well-PCR plate 50pL diluted enzyme sample were mixed with 50 pL sample buffer containing 2.7 mg DTT. The plate was sealed by Microseal ‘B’ Film from Bio-Rad and was placed into PCR machine to be heated to 70°C for 10 minutes. After that the chamber was filled by running buffer, gel cassette was set. Then 10 pL of each sample and standard (0.125-1.00 mg / mL BSA) was loaded on the gel and 5 pL of the markers were loaded. After that the electrophoresis was run at 200 V for 45 min. Following electrophoresis, the gel was rinsed 3 times 5 min in water, then stained in Safestain overnight and finally destained in water. Then the gel was transferred to Imager. Image Lab software was used for calculation of intensity of each band. A calibration curve was made using BSA (Thermo Scientific, Catalogue #23208) and the amount of the target protein was determined by the band intensity and calibration curve. The protein quantification method was employed to prepare enzyme samples of used in subsequent Examples.

[0192] Example 5 - Mature polypeptide sequence of Fusarium verticillioides xylanase variant FveXyn4_l

[0193] The amino acid sequence of the predicted form of FveXyn4_l protein is set forth as SEQ ID No. 3. This was expected to be the active form of the enzyme. However, we surprisingly identified the mature and active form of FveXyn4_l protein to be identical with the sequence set forth in SEQ ID No. 2, likely due to the host generated post-translation modifications and endogenous activities during fermentation influencing cleavage of the signal peptide.

[0194] Thus, FveXyn4_l was unexpectedly found to be less proteolytically modified in the final broth at the end of fermentation as compared to the predicted variant shown in SEQ ID NO:3. The variant generated was verified using mass spectrometry as described in detail below. Thus, the final expressed variant was less processed in the N-terminus as compared to the predicted 25 amino acids cleavage by SignalP version 4.0, resulting in a final N-terminal truncation of 23 amino acids corresponding to a mature version of FveXyn4_l having the polypeptide sequence of SEQ ID NO:2. This mature variant of FveXyn4_l has been used in all examples discussed herein.

[0195] To derive the exact polypeptide sequence of the mature variant of FveXyn4_l, the protein band of the protein was cut from an SDS-PAGE gel and digested using three different enzymes (Trypsin, A-Chymotrypsin and Glu-C) to prepare the sample for mass spectrometry analysis. Trypsin hydrolyzes peptide bonds specifically at the carboxyl side of arginine I and lysine (K) residues except when a proline (P) is on the carboxyl side. A-Chymotrypsin hydrolyzes peptide bonds specifically at the carboxyl side of tyrosine (Y), phenylalanine (F), tryptophan (W) and leucine (L) except when a proline (P) is on the carboxyl side. Glu-C preferentially cleaves at the carboxyl side of glutamyl (E) in ammonium bicarbonate buffer pH 8, but also cleaves at the carboxyl side of aspartyl (D) if the hydrolysis is carried out in a phosphate buffer pH 8.

[0196] To detect the exact C-terminal, the protein of interest was prepared for analysis using IFF procedure for protein characterization (A2963), with one change using 40% 180-water in the digestion buffer. The proteolytic cleavage will hereby incorporate both 180-water and 160- water in the resulting peptides, which consequently will appear as doublets in MS spectra. The protein C-terminal though will only appear as a single peptide with 160- water since it is not cleaved but just the “last peptide” left of the protein. In this way the C-terminal is mapped using MS / MS analysis. To detect the exact protein N-terminal, the intact protein is labeled with acetylation of the N-terminal before proteolytic digestion (IFF A-manual 3448). Guanidination of lysine converts lysine to homoarginine and protects lysine (side chain) from being acetylated. Only the peptide originating from the protein N-terminal will be acetylated and hereby unambiguously identified.

[0197] Example 6 - Viscosity reduction and filterability of rye-based mashing trials with different xylanases in combination with a-L-Arabinofuranosidase and Feruloyl esterase

[0198] The objective of this example was to demonstrate the benefit of having an un-inhibited xylanase present during a 100% rye based mashing process. Enzymes was tested in a mashing operation model system for wort production using milled Rye (Supplied by Hiram Walker, US., Material Code: 202109-2201) milled at a Buhler Miag malt mill 0.5 mm setting, position 4).

[0199] Mashing operation for wort production

[0200] Rye grist (60.0g milled Rye grains) was mixed in beakers with 203g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid., resulting in a water to grist ratio of 3.38:1.

[0201] The FveXyn4_l xylanase variant was added based on mg protein determined according to example 4 in the following setup:

[0202] Trial 1, 1,89 CMC-DNS U PfunXyn per g Rye; Trial 2, 6.8 pg FveXyn4_l protein per g Rye; Trial 3, 1,89 CMC-DNS U PfunXyn per g Rye + 0.3 U AniAfur per g Rye + 0.3 U BaAfur per g Rye; Trial 4, 1,89 CMC-DNS U PfunXyn per g Rye + 0.3 U AniAfur per g Rye + 0.3 U CtFe per g Rye; Trial 5, 1,89 CMC-DNS U PfunXyn per g Rye + 0.3 U BaAfur per g Rye + 0.3 U CtFe per g Rye Trial 6, 6.8 pg FveXyn4_l protein per g Rye + 0.3 U AniAfur per g Rye + 0.3 U BaAfur per g Rye; Trial 7, 6.8 pg FvcXyn4_l protein per g Rye + 0.3 U AniAfur per g Rye + 0.3 U CtFe per g Rye; Trial 8, 6.8 pg FveXyn4_l protein per g Rye + 0.3 U BaAfur per g Rye + 0.3 U CtFe per g Rye. In addition to enable generation of fermentable sugars and appropriate FAN levels the 0.730 mg AMYLEX® 6T per g rye grist was added to each trial. The grist was mashed with the program; heated to 45°C and kept for 40 minutes for mashing in; heated to 65°C for 10.0 minutes by increasing temperature with l°C / minute; kept at 65°C for 40 minutes; heated to 74°C for 10 minutes by increasing temperature with l°C / minute; kept at 74°C for 160 minutes and mashing off.

[0203] Hereafter, iodine negative was tested when temperature had reached 74°C and mashed off. The time in minutes that was required to get iodine negative was noted and result are given in table 2. It was here clear that the un-inhibited FveXyn4_l by a lower dosage and in shorter time were able to obtain an iodine negative result of the mash as compared to PfunXyn, indicating a further processed starch fraction of the Rye material.

[0204] Table 2, Iodine testing of Rye mashing. The time in minutes that was required to get iodine negative is noted by OK for trial 1-2 with the following alpha-amylase addition: Trial 1, 6.8 pg FveXyn4_l protein per g Rye and Trial 2, 1,89 CMC-DNS U PfunXyn per g rye.

[0205] After mashing off each beaker was adjusted to 263 g with tap water and cooled to 25°C before centrifugation. Separation was performed by centrifugation; 4000 rpm for 5 minutes (5°C) in swing bucket centrifuge (Thermo Scientific SL 8R Centrifuge). The volumes of supernatant were measured in mL (measuring glass) after centrifugation and result are given in table 3. It is clear’ that the un-inhibited FveXyn4_l resulted in much larger supernatant volume, being equivalent to an increased filtration yield. Table 3, Mash supernatant volume after centrifugation. Trial 1 , 6.8 pg FveXyn4_l protein per g Rye and Trial 2, 1,89 CMC-DNS U PfunXyn per g rye.

[0206] Turbidity of the mash supernatant samples after mashing was measured using an Anton Paar (DMA 5000, HazeQC ME module) according to Dupont Standard Instruction Brewing, 23.8580-B28 and shown very different depending on the xylanase use in experiments. The turbidity (S90 / S25 EBC) was measured at a 90° scatter angle to detect the presence of small particles and turbidity measured at 25° scatter angle was included as additional information on larger particles. The turbidity data were shown in Figure 1 and 2 with S90 and S25 turbidity respectively. The three samples: Trial 3, 4 and 5, all with PfunXyn had significant higher haze values than the corresponding samples 6, 7, and 8 with FveXyn4_l. Thus, the haze data were in accordance with the difference observed in supernatant volume all the samples from the series with PfunXyn resulted in higher turbidity than the samples from the series with FveXyn4_l, suggesting effective lowering of particles in solution by FveXyn4_l. This was further found independent on the two a-L-Arabinofuranosidases and the Feruloyl esterase added as accessory enzymes together with the xylanase. These results clearly demonstrate the superior properties of FveXyn4_l for Rye arabinoxylan breakdown.

[0207] Specific gravity, density and viscosity was determined on the supernatant of Rye mash samples using a DMA 5000 Density meter and Lovis 2000 M / ME rolling-ball viscometer (both from Anton Paar. Gratz. Austria).

[0208] Original Extract (OE) Extract in the samples after mashing was measured using an Anton Paar (DMA 5000) following Dupont Standard Instruction Brewing. 23.8580-B28. The results are shown in table 4.

[0209] From the data in Table 4 it was observed that data was not given for all samples, simply due to too high viscosity. The extract content seemed to be very similar for all samples meaning that the application of arabinofuranosidases and / or feruloyl esterase did not give rise to increases in extract, e.g., water unextractable extract did not become extractable. The extract contents were as well similar in samples for applying cither PfunXyn or FvcXyn4_l, with slight tendency that sample with FveXyn4_l showed higher extract together with arabinofuranosidases and / or feruloyl esterase. The density and specific gravity were found to be very similar in all samples. Its apparent from the viscosity data as determined by the Anton Paar that mash viscosity was lower when FveXyn4_lwas applied and with less variation in viscosity from sample to sample than for the samples that were applied PfunXyn.

[0210] Table 4, Density (g / cm3), Specific Gravity (SG), original extract and viscosity for supernatant of

[0211] Rye mashes with xylanase, a-L-Arabinofuranosidase (Afur) and Feruloyl esterase (CtFe).

[0212] In addition to characterization by Anton Paar of mashes, shear (flow) viscosity of samples were measured at 20°C using an Anton Paar rheometer type MCR301 or MCR302e with measuring system “Double Gap (DG26.7)”. The viscosity was measured over shear rates from 0.1 to 100 s-1 using a logarithmic ramp with integration times from 20 to 1 s. Viscosity results was obtained at the specific shear rate at 10 s-1 and are shown in table 5.

[0213] Table 5, Flow viscosity determined for supernatant from Rye mashes with xylanase, a-L- Arabinofuranosidase (Afur) and Feruloyl esterase (CtFe). The viscosity was read at shear rate 10 s-1 at 20°C.

[0214] The viscosity reduction with FveXyn4_l was higher when used on its own that the achieved viscosity reduction when PfunXyn was applied in combination with one or both arabinofuranosidases. This suggested that FveXyn4_l did not have the same requirement of getting the single and the double substituted arabinofuranoses unit present in Rye arabinoxylan cut off for being able to hydrolyze the xylan backbone of the arabinoxylan. This could mean that PfunXyn required longer unsubstituted xylan backbone for making the hydrolysis happen than FveXyn4_l. In other words, FveXyn4_l may be able to hydrolyze xylan with higher degree of substituted arabinofuranose.

[0215] Example 7 -Application of xylanases for Rye mashing in brew analysis with determination of real degree of fermentation (RDF) and alcohol content Rye mashes were prepared similar to example 6 with following differences. Rye grist (65.0g milled Rye grains) was mixed in beakers with 220g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid., resulting in a water to grist ratio of 3.38:1.

[0216] The FveXyn4_l xylanase variant was added based on mg protein determined according to example 4 in the following setup:

[0217] Trial 1 to 3, 1,89 CMC-DNS U PfunXyn per g Rye; Trial 4 to 6, 6.8 pg FveXyn4_l protein per g Rye. In addition to enable generation of fermentable sugars and appropriate FAN levels the 0.184 mg AMYLEX® 6T per g rye grist was added to at mashing in 66°C and 0.184 mg AMYLEX® 6T per g rye grist was added to main mashing in 74°C at each trial. The grist was mashed with the program; heated to 66°C for mashing in and pH adjustment before enzymes were applied and hereafter kept at 66°C for 40 minutes; heated to 74°C for 8.0 minutes by increasing temperature with l°C / minute; kept at 74°C for 55 minutes; second dose of AMYLEX® 6T and kept at 74°C for another 105 minutes; content of each sample was adjusted to 285g; cool to 35°C. 220 g of each mash was weighed into a 500 mL conical flask for fermentation adding 15 mL of yeast slurry (0.5% yeast, SafSpirit HG-1 dry yeast for distilling from Fermentis) to the mash having 35 °C; mash was fermented on the grain.

[0218] The remaining of the mash was separated by centrifugation; 4000 rpm for 5 minutes (5°C) in swing bucket centrifuge (Thermo SCIENTIFIC SL 8R Centrifuge). The supernatant was used for analysis, see below. The samples were fermented at 35°C and 150 rpm after yeast addition. All sample were added 0.262 mg DIAZYME® 480 per g rye to facilitate saccharification during fermentation. Analysis was performed when fermentation had finished. Supernatant analysis: Original Extract (OE), Extract in the wort samples after mashing was measured using Anton Paar (Lovis) following Standard Instruction Brewing, 23.8580-B28. The content of Free Alpha- Amino Nitrogen (mg / litre) (FAN) was measured in the wort following Standard Instruction Brewing, 23.8580-B15 using Spectrophotometer Genesys 10S UV-Vis (Based on EBC 8.10).

[0219] Beer analysis: RDF was measured using an Anton Paar (DMA 5000) following Standard Instruction Brewing, 23.8580-B28 and alcohol by Standard Instruction Brewing, 23.8580-B28. Real degree of fermentation (RDF) value may be calculated according to the equation below:

[0220] Where: RE = real extract = (0.1808 x °Pinitiai) + (0.8192 x0Pfinai), °Pinitiai is the specific gravity of the standardised worts before fermentation and °Pfinaiis the specific gravity of the fermented worts expressed in degree Plato.

[0221] In the present context, Real degree of fermentation (RDF) was determined from the specific gravity and alcohol concentration.

[0222] Specific gravity and alcohol concentration was determined on the fermented samples using a Beer Alcolyzer Plus and a DMA 5000 Density meter (both from Anton Paar, Gratz, Austria). Based on these measurements, the real degree of fermentation (RDF) value was calculated according to the equation below:

[0223] Where: E(r) is the real extract in degree Plato (°P) and OE is the original extract in °P.

[0224] Original Extract (OE) Extract in the beer samples after mashing was measured using an Anton Paar (DMA 5000) following Standard Instruction Brewing, 23.8580-B28. Alcohol By Volume (%V / V) The achieved Alcohol By Volume (ABV) was measured using an Anton Paar (DMA 5000) following Standard Instruction Brewing, 23.8580-B28.

[0225] Real degree of fermentation (RDF), extract and alcohol content of the fermented beer are shown in table 6. Both the obtained RDF values, extract and alcohol values were higher of fermented beer with FveXyn4_l applied in mashing as compared to PfunXyn. The extract average of three ferments with PfunXyn was 16.71°P and three ferments with FveXyn4_lwas 17,04°P. The RDF average of three ferments with PfunXyn was 77.50% and three ferments with FveXyn4_lwas 77.58%. The alcohol content average of three ferments with PfunXyn was 8.70% and three ferments with FveXyn4_lwas 8.89%.

[0226] It is clear that the increased arabinoxylan solubilization during mashing also increased RDF and alcohol content in the fermented Rye mash with FveXyn4_l included. Table 6, Extract, RDF and alcohol content after beer fermentation of wort produced with different xylanascs.

[0227] Example 8 - Arabinoxylane analysis of Rye wort samples prepared with different xylanases in combination with a-L-Arabinofuranosidase and Feruloyl esterase.

[0228] The content of soluble arabinoxylans was quantified in the Rye mash produced as described in example 6. LC-MS reverse phase chromatography was used to characterize and quantify the content of the different soluble arabinoxylan were fractionated by DP (extending from AXl / mono to AX7+) on an Agilent 1290 BinPump system coupled to a Bruker maXis QTOF 4G. The chromatographic separations was performed on a Acquity UPEC® 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) MethanokTB A: Acetic Acid 1000:2.4:10 (B) gradient run for 16 min, Injection volume 10 pl, flow rate of 200 pl / min. The effluent from the column was coupled via an clcctrospray ionisation interface (Ionisation: ESI (electrospray) in Negativ mode) to the high-resolution maXis mass spectrometer. The following spectrophotometer setting was applied: Nebulizer: 2.0 Bar, dry gas: 8 E / min and dry heater: 200 °C.

[0229] The relative distribution of soluble AX components in rye mash prepared with xylanase together with a-L-Arabinofuranosidase and Feruloyl esterase are shown in Table 7. HyStar LC with Agilent ICF software was used for quantification. For identification of soluble AX components, the following pentose standards was applied: Xylose, xylobiose, xylotriose, xylotetraose, xylypentaose, xylohexaose and xyloheptaose. AX fractions were determined for the two samples applied cither PfunXyn or FvcXyn4_l with combinations of a-L- Arabinofuranosidase and Feruloyl esterase. The method was limited to determine the soluble arabinoxylan which means AX9 or lower.

[0230] The data clearly shows that the proportions of AX2 and AX3 are significantly higher and the proportions of AX4 to AX7 were lower for the sample applied PfunXyn. Thus, FveXyn4_l clear demonstrate increased specificity for larger soluble dextrin generation supporting more efficient high molecular AX components turnover and thereby lowering of solution viscosity.

[0231] Table 7, the relative distribution of arabinoxylans (AX) by DP in Rye mash prepare with xylanase together with a-L-Arabinofuranosidase and Feruloyl esterase.

[0232] Example 9 - Xylanase rye and wheat inhibition assay.

[0233] One factor limiting the efficiency of xylanase action on various cereals may be the presence of specific proteinaceous inhibitors of xylanases in grains. Proteinaceous soluble inhibitors of xylanases been found in barley, rye, com and other cereals. The example describes a simple and novel sensitivity assay to rye and wheat xylanase-inhibitor-protein-like (XIP-like) inhibitors. The inhibition degree of rye and wheat was determined for various xylanase products. Rye and wheat XIP inhibitor extracts were prepared by the following procedure. A weighted amount of either rye or wheat flour (Valsempllen, Denmark) (20 g) was suspended in 200 mL (10% w / v) of 0.1 M Na-acetate buffer, pH 5.6, and the mixture agitated on a shaker at 300 rpm for 3.0 hr. The insoluble portion was separated by centrifugation at 5,000 rpm for 20 min in a swing-bucket centrifuge. The supernatants were treated as Rye and Wheat XIP inhibitor extracts pH 5.6 respectively. Dilution series ranging from 500 - 1000000 times, were prepared 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 with the following xylanase samples; BsbBglu + AkaXyl_var, TrXyl, PfunXyn, TemXyn and FveXyn4_l all obtained from Dupont Nutrition Bioscience Aps, Denmark, Shearzyme P105, Viscoferm and Ultraflo Max from Novozymes, Denmark. All combinations of diluted enzyme samples were incubated 24 hours at 5°C to facilitate potential inhibition and following 1 mL 0.1 M Na-acetate buffer, pH 5.6 was mixed with 1 mL of the diluted enzyme sample in a reaction tube and placed 5 min at 40°C using a water bath for equilibration. One Xylazyme tab purchased from Megazyme (Xylazyme, T-XYZ-200T to assay endo-l,4-b-Xylanase activity from Megazyme, Ireland) was added to each reaction tube, incubated 10 minutes at 40°C and all reactions were stopped by addition of 10 mL demineralized water according to manufactures declaration. The reaction solution was filtered by paper filter (Whatman, Cytiva Grade 597 Plus Qualitative Filter Paper, Circles) and absorbance at 590nm was determined using a Spectrophotometer (Genesys Spectrophotometer 10S UV-Vis). The %-Rye XIP and %-Wheat XIP inhibition were calculated using the following formulas below, only using the enzyme sample dilution resulting in absorbance values ranging between 0.0 and 1.0: % Rye XIP inhibition

[0234] Abs 590 nm (enzyme diluted in buffer) — Abs 590nm (enzyme diluted in rye extract) Abs 590 nm (enzyme diluted in buffer)

[0235] Or

[0236] % Wheat XIP inhibition

[0237] Abs 590 nm (enzyme diluted in buffer) — A Abs 590 nm (enzym

[0238] The result of testing rye and wheat XIP inhibition of the various xylanase samples are shown in table 8. There was surprisingly very large difference found and FveXyn4_l clearly showed very little XIP inhibition, especially when incubated with rye (17.9 % inhibition), however also in the presence of wheat (51.1% inhibition), which may explain the superior viscosity reduction obtained in Rye mashes of FveXyn4_l. Out of all tested xylanase samples, FveXyn4_l showed superior low Rye XIP inhibition.

[0239] Table 8, Xylanase activity (Xylazyme, Megazyme) after incubation 24 hours 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. The %-Rye and %- Wheat XIP inhibition is calculated to formulas described in example 9.

[0240] Example 10 - Rye mashing trials with dose-response test of inhibited and un-inhibited xylanases and their effect on viscosity reduction and filterability.

[0241] The objective of this example was to test potential benefits of a Rye XIP un-inhibited vs inhibited xylanase present during a 100% rye based mashing process, using large dosage range. Enzymes was tested in a mashing operation model system for wort production using milled Rye (Supplied by Hiram Walker, US., Material Code: 202109-2201) milled at a Buhler Miag malt mill 0.5 mm setting, position 4). As an example of a Rye XIP un-inhibited xylanase, FveXyn4_l (17.9% inhibition) was applied and as an example of a Rye XIP inhibited xylanase PfunXyn (84.9% inhibition) was applied, according to Rye XIP inhibition as described in example 9.

[0242] Mashing operation for wort production

[0243] Rye grist (60.0g milled Rye grains) was mixed in beakers with 203g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid., resulting in a water to grist ratio of 3.38:1. The FveXyn4_l xylanase variant was added based on mg protein determined according to example 4. PfunXyn was dosed 0.787 to 15.7 CMC-DNS U per g Rye and FveXyn4_l dosed 5.7 to 113.3 g FveXyn4_l protein per g Rye for comparison. In all trials 0.368 mg AMYEEX® 6T per g rye grist was added to at mashing in 66°C to enable generation of fermentable sugars and appropriate FAN levels. The grist was mashed with the program; heated to 45 °C for mashing in and pH adjustment before enzymes were applied and hereafter kept at 45°C for 40 minutes; heated to 65°C for 20 minutes by increasing temperature with l°C / minute; kept at 65°C for 40 minutes; heated to 74°C for 9 minutes by increasing temperature with l°C / minute; and kept at 74°C for another 160 minutes; content of each sample was adjusted to 263g; cooled to 25°C before centrifugation.

[0244] Mash separation was performed by centrifugation; 4000 rpm for 5 minutes (5°C) in swing bucket centrifuge (Thermo SCIENTIFIC SE 8R Centrifuge). The volumes of the wort were measured in mF (measuring glass) after centrifugation. The wort samples were boiled for 60 minutes in a boiling bath and samples were cooled down to 17°C. All the samples were filtered and used for analysis, sec below.

[0245] Wort sample extract, density and flow viscosity were determined as described in example 6 and the results are shown in table 9 and 10.

[0246] The density and specific gravity were found to be very similar in all samples. Minor increase in original extract was observed by increased dosage for both xylanase samples.

[0247] Surprisingly, it was apparent from the viscosity data as determined that mash viscosity was lower when FveXyn4_l was applied in broad range of dosages and with less variation in viscosity from sample to sample than for the samples that were applied PfunXyn. All dosages of FveXyn4_l resulted in lower mash viscosity as compared to any applied dose of PfunXyn. In addition, as see in table 9 the volume of supernatant collected from mash treatments was clearly larger for all dosages of FveXyn4_l as compared to PfunXyn, hereby enabling higher yield. Table 9, Wort sample Density (g / cm3), Specific Gravity (SG), Original extract and flow viscosity (read at shear rate 10 s-1 at 20°C) determined for Rye mashes with the xylanases PfunXyn and FveXyn4_l in various dosages as indicated.

[0248] Table 10, Volume collected after centrifugation of Rye mashes with the xylanases PfunXynand FveXyn4_l in various dosages as indicated.

[0249] Example 11 - Malt mashing trials with dose-response test of Rye inhibited and un-inhibited xylanases and their effect on viscosity reduction and filterability.

[0250] The objective of this example was to test a Rye XIP un-inhibited vs inhibited xylanase present during a 100% malt based mashing process, using various dosages. Enzymes was tested in a mashing operation model system for wort production using milled Malt (Pilsner Malt from Fuglsang, Denmark, Batch Number 12.10.2019) at a Buhler Miag malt mill 0.5 mm setting, position 4). As an example of a Rye XIP un-inhibited xylanase, FveXyn4_l (17.9% inhibition) was applied and as an example of a Rye XIP inhibited xylanase PfunXyn (84.9% inhibition) was applied, according to Rye XIP inhibition as described in example 9. Mashing operation for wort production

[0251] Malt grist (60.0g milled Malt grains) was mixed in beakers with 203g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid., resulting in a water to grist ratio of 3.38:1. The FveXyn4_l xylanase variant was added based on mg protein determined according to example 4. PfunXyn was dosed 0.315 to 1.570 CMC-DNS U per g Rye and FveXyn4_l dosed 2.27 to 11.3 pg FveXyn4_l protein per g Malt for comparison. The grist was mashed as described in example 10 also regarding mash separation and following mash analysis.

[0252] Wort sample extract, density and flow viscosity were determined as described in example 6 and the results are shown in table 11 and 12.

[0253] The density, specific gravity and original extract were found to be very similar in all samples, generated by using both xylanase samples. In addition, no significant differences were observed in mash viscosity for the Malt mashes in contrast to Rye. The volume of supernatant collected from mash treatments as see in table 12 was identical for all dosages of FveXyn4_l and PfunXyn applied, hereby indicating no effect applying a Rye XIP un-inhibited vs inhibited xylanase in malt mashes.

[0254] Table 11, Wort sample Density (g / cm3), Specific Gravity (SG), Original extract and flow viscosity (read at shear rate 10 s-1 at 20°C) determined for Malt mashes with the xylanases PfunXyn and FveXyn4_l in various dosages as indicated.

[0255] Table 12, Volume collected after centrifugation of Malt mashes with the xylanases PfunXyn and FveXyn4_l in various dosages as indicated.

[0256] Example 12 - Performance of xylanases in Malt mashing trials with high Rye inclusion

[0257] The objective of this example was to test a Rye XIP un-inhibited vs inhibited xylanase present during a 50% malt - 50% rye based mashing process, using various xylanase dosages. Enzymes was tested in a mashing operation model system for wort production using milled Malt (Pilsner Malt from Sophus Fuglsang, Denmark, Batch Number 10.12.2019) at a Buhler Miag malt mill 0.5 mm setting, position 4) and milled Rye (Wey Rye 3-8 EBC from Maltbazaren, Denmark, Batch: 12.03.2020, DK20-00066) milled at a Buhler Miag malt mill 0.5 mm setting, position 4).

[0258] As an example of a Rye XIP un-inhibited xylanase, FveXyn4_l (17.9% inhibition) was applied and as an example of a Rye XIP inhibited xylanase PfunXyn (84.9% inhibition) was applied as well as TemXyn (40.0% inhibition), according to Rye XIP inhibition as described in example 9.

[0259] Mashing operation for wort production Rye grist (35.0g milled Rye grains) and Malt grist (35.0g milled Malt grains) were mixed in beakers with 210g of tap water in mashing bath (Lockncr, LG-clcctronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid, resulting in a water to grist ratio of 3.1:1. The FveXyn4_l xylanase variant was added based on mg protein determined according to example 4. PfunXyn was dosed 3.15 to 9.45 CMC-DNS U per g Grist, TemXyn was dosed 5.52 to 16.57 U per g Grist and FveXyn4_l dosed 22.7 to 68.0 pg FveXyn4_l protein per g Grist for comparison. The grist was mashed with the program; heated to 52°C for mashing in and pH adjustment before enzymes were applied and hereafter kept at 52°C for 10 minutes; heated to 65 °C for 13 minutes by increasing temperature with l°C / minute; kept at 65 °C for 45 minutes; heated to 72°C for 7 minutes by increasing temperature with l°C / minute; and kept at 72°C for another 15 minutes; heated to 78°C for 6 minutes by increasing temperature with l°C / minute; and kept at 78 °C for another 30 minutes - mashing off; content of each sample was adjusted to 350g and filtered. The volumes of the wort collected after 5, 10, 15 and 30 minutes were measured, and pH was adjusted to pH 5.2 with 2.5 M sulphuric acid. One pellet of hops was added to each flask. The wort samples were boiled for 60 minutes in a boiling bath and samples were cooled down to 17°C. All the samples were filtered used for analysis, see below.

[0260] Wort sample extract, density and flow viscosity were determined as described in example 6 and the results are shown in table 13 and 14.

[0261] The density and specific gravity were found to be very similar in all samples. Minor increase in Real extract was observed in some samples by FveXyn4_l xylanase.

[0262] Surprisingly, it was apparent from the viscosity data as determined that mash viscosity was lower when FveXyn4_l was applied in broad range of dosages and with less variation in viscosity from sample to sample than for the samples that were applied PfunXyn and TemXyn. All dosages of FveXyn4_l resulted in lower mash viscosity as compared to any applied dose of PfunXyn and TemXyn. In addition, as see in table 14 the turbidity of Malt-Rye wort samples determined as haze by EBC scattering at 25 and 90 degrees by Anton Paar at 20°C clearly demonstrated that wort samples prepared with FveXyn4_l resulted in much clear samples with less turbidity or haze as compared to wort pared with either PfunXyn or TemXyn, respectively.

[0263] In addition, as see in table 15 the volume of supernatant collected from mash filtration was clearly larger for all dosages of FveXyn4_l as compared to PfunXyn and TemXyn for all applied dosages, clearly enabling higher yield treating a 50% malt - 50% Rye mash. Tabic 13, Wort sample Density (g / cm3), Specific Gravity (SG), Real extract and Anton Paar viscosity (12 °P) determined for Malt-Rye mashes with the xylanases PfunXyn, TemXyn and FveXyn4_l in various dosages as indicated.

[0264] Tabic 14, Turbidity of Malt-Rye wort samples determined as haze by EBC scattering at 25 degree (S25 / S0) or 90 degrees (S90 / S0) by Anton Paar at 20°C for mashes with the xylanases PfunXyn, TemXyn and FveXyn4_l in various dosages as indicated.

[0265]

[0266] Table 15, Filtration volume collected after 5, 10, 15 and 30 min of mash filtration (paper filter) of 50% Malt- 50% Rye mashes with the xylanases: PfunXyn, TemXyn and FveXyn4_l in various dosages as indicated.

[0267]

[0268] Example 13 - Comparison of xylanase performance in Malt mashing trials with high Rye, Wheat and Barley inclusion

[0269] The objective of this example was to test a Rye XIP un-inhibited vs inhibited xylanase present during a 60% malt: 40% rye, a 60% malt: 40% wheat and a 60% malt: 40% barley based mashing process, using various xylanase dosages. Enzymes was tested in a mashing operation model system for wort production using milled Malt (Pilsner Malt from Sophus Fuglsang, Denmark, Batch Number 10.12.2019) at 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 at a Buhler Miag malt mill 0.5 mm setting, position 4, milled barley (Raw Barley from Sophus Fuglsang, Denmark, Batch Number 09.03.2019) at a Buhler Miag malt mill 0.5 mm setting, position 4 and milled wheat (Wheat Whole kernel organic from XXX, Batch Number 17.11.2022-1) at a Buhler Miag malt mill 0.5 mm setting, position 4.

[0270] As an example of a Rye XIP un-inhibited xylanase, FveXyn4_l (17.9% inhibition) was applied and as an example of a Rye XIP inhibited xylanase was TemXyn (40.0% inhibition) applied as well as, according to Rye XIP inhibition as described in example 9. Mashing operation for wort production

[0271] Rye, Wheat or Barley grist (28.0g milled Rye grains) and Malt grist (42.0g milled Malt grains) were mixed in beakers with 210g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid, resulting in a water to grist ratio of 3.1:1. The FveXyn4_l xylanase variant was added based on mg protein determined according to example 4. TemXyn was dosed 0.27 to 0.69 U per g Grist and FveXyn4_l dosed 9.0 to 27.2 pg FveXyn4_l protein per g Grist for comparison. To all trials BsdBglu was added to support the xylanase in a varying dose of 2-5U / kg grist, identical for both xylanases used.

[0272] The grist was mashed with the program; heated to 60°C for mashing in and pH adjustment before enzymes were applied and hereafter kept at 60°C for 5 minutes; heated to 65°C for 5 minutes by increasing temperature with l°C / minute; kept at 65°C for 45 minutes; heated to 72°C for 7 minutes by increasing temperature with l°C / minute; and kept at 72°C for another 20 minutes; heated to 78°C for 6 minutes by increasing temperature with l°C / minute; and kept at 78°C for another 10 minutes - mashing off; content of each sample was adjusted to 350g and filtered. The volumes of the wort collected after 5, 10, 15 and 30 minutes were measured (Figure 3, results shown as an average over all dosages for each xylanase), and pH was adjusted to pH 5.2 with 2.5 M sulphuric acid. One pellet of hops was added to each flask. The wort samples were boiled for 60 minutes in a boiling bath and samples were cooled down to 17°C. All the samples were filtered used for analysis, see below.

[0273] Wort sample extract and viscosity were determined as described in example 6 and the results shown as an average over all dosages (TemXyn was dosed 0.27 to 0.69 U per g Grist and FveXyn4_l dosed 9.0 to 27.2 pg FveXyn4_l protein per g Grist) for each xylanase are presented in figure 4 and 5.

[0274] It can be observed that the average wort volume collected after 30 minutes filtration is increased using FveXyn4_l as compared to TemXyn over range of xylanase dosages (Figure 3). Furthermore, it’s clear that the relative increase is highest for mash composed of 40:60 ryemralt, followed by 40:60 wheatmialt and smallest for 40:60 bariey:malt.

[0275] In figure 4 is seen that the average wort Viscosity at 12°P (mPaxs) after filtration is decreased using FveXyn4_l as compared to TemXyn over range of xylanase dosages. The relative largest decrease is observed for mash composed of 40:60 rye:malt, followed by 40:60 whcat:malt and smallest for 40:60 barlcy:malt.

[0276] In figure 5 is seen that the average Original Extract (°P) of wort after filtration is increased using FveXyn4_l as compared to TemXyn over range of xylanase dosages. The relative increase are largest for mash composed of 40:60 rye:malt and 40:60 wheat:malt and only minor for 40:60 barley:malt.

[0277] The benefits of using the Rye XIP un-inhibited xylanase, FveXyn4_l in terms of increased filtration volume or rates by lower viscosity and increased Original Extract (°P) are clearly most significant using Rye or Wheat in mash as compared to malt or barley, in correspondence with less XIP inhibition for these raw materials.

[0278] Example 14 - Effect of xylanase on ethanol production in Rye-Malt SSF trials with high Rye inclusion.

[0279] The objective of this example was to test a Rye XIP un-inhibited vs inhibited xylanase present during a rye based mashing and fermentation process, using various xylanase dosages. Enzymes was tested in a mashing operation model system for wort production using milled Malt (Distiller’s malt: DK19-01017, Denmark) at a Buhler Miag malt mill 0.5 mm setting, position 4) and milled Rye (Briess Raw rye: Denmark, Batch: DK 20-00053) milled at a Buhler Miag malt mill 0.5 mm setting, position 4). As an example of a Rye XIP un-inhibited xylanase, FveXyn4_l (17.9% inhibition) was applied and as an example of a Rye XIP inhibited xylanase PfunXyn (84.9% inhibition) was applied as well as Viscoferm (41.3% inhibition), according to Rye XIP inhibition as described in example 9.

[0280] Mash protocol no enzyme addition

[0281] All raw materials were milled at setting #4 and mixed with water with a mash temperature of 55°C. Adjunct Liquefaction: Temperature was adjusted to 55°C for 1 minute for mashing in, the total grist of 66.5 g rye + 1.75 g malt was added to 210.0 grams water. pH adjustment to 5.5 if needed. No enzyme addition. Temperature was increased to 95°C with l°C / minute and held for 40 minutes. Saccharification was tested with iodine test at 10, 20, and 30 min at 95°C. Main Mashing: Temperature for mashing in was adjusted to 65°C. Thus, the mash was cooled to 65°C and added conversion 1.75 g malt. Mash was kept at 65°C for 30 minutes and saccharification of starch was tested with iodine after 30 minutes. Then cooled to 32°C, kept for 5 minutes and each beaker was adjusted to 280 g with tap water. The mash was filled into a 500- ml conical flask for fermentation adding 0.5 % yeast (1.4 g for 280 g mash) to the wort having 32°C / 90°F. Then yeast (White Labs Distilling Yeast: DK 202009-0301) was added for fermentation. No enzyme was added, and Mash was fermented on the grain. Temperature was set to 35°C / 95°F and the fermentation lasted for 3 days.

[0282] Mash protocol with enzyme addition

[0283] Adjunct Liquefaction: Temperature was adjusted to 55°C for 1 minute for mashing in, the total grist of 66.5 g rye + 1.75 g malt was added to 210.0 grams water. pH adjustment to 5.5 if needed. After correct pH was measured, alpha amylase (0.15 kg / t grist) was added to ensure efficient liquefaction (AMYLEX 5T, 1.050g dissolved in lOOmL provided from Dupont Nutrition Bioscience Aps Denmark). Temperature was increased to 95 °C with l°C / minute and held for 40 minutes. Saccharification was tested with iodine test at 10, 20, and 30 min at 95°C. Main Mashing: Temperature for mashing in was adjusted to 65°C. Xylanase was dosed on g sample / kg grist according to table 16 below. Thus, the mash was cooled to 65 °C and added conversion 1.75 g malt. Mash was kept at 65°C for 30 minutes and saccharification of starch was tested with iodine after 30 minutes. Then cooled to 32°C, kept for 5 minutes and each beaker was adjusted to 280 g with tap water.

[0284] The mash was filled into a 500-ml conical flask for fermentation adding 0.5 % yeast (1.4 g for 280 g mash) (White Labs Distilling Yeast: DK 202009-0301) to the wort having 32°C / 90°F. For all trials a glucoamylase (0.8 kg / t grist) was added to ensure efficient saccharification (Diazyme SSF2, 1.750g dissolved in lOOmL provided from Dupont Nutrition Bioscience Aps Denmark). Mash was fermented on the grain. Temperature was set to 35°C / 95°F and the fermentation lasted for 3 days.

[0285] Table 16, Trial setup with addition raw material in Liquifaction 95°C and Main Mashing 65°C including xylanase addition (Viscoferm, PfunXyn and FveXyn4_l).

[0286]

[0287] Wort sample density, specific gravity and viscosity were determined as described in example 6 and the results are shown in table 17. The density and specific gravity were found to be very similar in all samples, viscosity of the control was simply too high to enable analysis.

[0288] Again, it was apparent from the viscosity data as determined that mash viscosity was lower when FveXyn4_l was applied in broad range of dosages and with less variation in viscosity from sample to sample than for the samples that were applied PfunXyn and Viscoferm. All dosages of FveXyn4_l resulted in lower mash viscosity as compared to any applied dose of PfunXyn and TemXyn.

[0289] Table 17, Wort sample Density (g / cm3), Specific Gravity (SG), Real extract and Anton Paar viscosity (12 °P) determined for Malt- Rye mashes with the xylanases Viscoferm, PfunXyn and FvcXyn4_l in various dosages as indicated.

[0290]

[0291] Real Degree of Fermentation was measured using an Anton Paar (DMA 5000) following Standard Instruction Brewing, 23.8580-B28 and Alcohol By Volume (%V / V) (ABV) was measured using an Anton Paar (DMA 5000) following Standard Instruction Brewing, 23.8580- B28.

[0292] The effect of an increasing dose rate of xylanase on ethanol yield was also looked at. FveXyn4_l provided the highest levels of ethanol in addition to having the lowest viscosity as seen on figure 6. The results clearly showed that an increasing dose rate of FveXyn4_l also resulted in increasing ethanol yields and more than what was obtained by PfunXyn and FveXyn4_l in a rye-based SSF fermentation.

[0293] Example 15 - Xylanase thermostability in malt-based wort

[0294] Xylanase thermostability was tested in malt-based wort

[0295] A 100% malt-based wort was prepared from malt extract as follows: 1100 g Munton's Light Malt Extract (Batch XB 35189, expiry date 01-2024) was dissolved in 3000 ml warm tap water (45°C). This slurry was stirred for about 10 min until the liquid was homogeneous and the pH was adjusted to 5.6 with 2.5 M sulphuric acid. To the slurry was added 10 pellets of Bitter hops from Hopfenveredlung, St. Johann, Germany: Alpha content of 16,0 % (EBC 7.7 0 specific HPLC analysis, 01.10.2023), then split in 500mL blue-cap bottles and boiled for 1 hour to ensure

[0296] 16 protein precipitation and avoid potential microbial contamination. The final wort had an initial Specific Gravity of 1048 (i.c. 12 °Plato). 200g of the filtered wort was added to a 500 ml and frozen until use.

[0297] The xylanase activity was determined colorimetricly by monitoring the rate of degradation of 4,6-O-(3-Ketobutylidene)-4-nitrophenyl-0-D-45-glucosyl-xylopentaoside substrate (XylX6). The release of the substrate's 4-nitrophenol directly related to the hydrolysis of the XylX6 by the endo-xylanase activity and is measured at 405nm. The colorimetric substrate contained in the XylX6 assay kit (K-XylX6, Megazymes) is combined with a 0- xylosidase which allows for the release of the colorimetric group 4-nitrophenol. The blocked XylX6 substrate exhibits the distinct advantage over commonly employed colorimetric oligosaccharide substrates in that the ketobutylidene acetal on the non-reducing terminal residue acts as a “blocking group” preventing hydrolysis by exo-acting enzymes including b-xylosidase and b-glucosidase which commonly occur in crude sample extracts. The XylX6 Substrate used was “Bottle 1” of Megazyme kit K-XylX6-2V purchased from Megazyme Ireland. 0,1 M Sodium Acetate Buffer pH 5.0 (for Substrate Prep only) was prepared by dissolving 9.57g of Sodium Acetate (trihydrate) in 800 mL of Milli-Q water. Adjustment of pH to 5.0 with concentrated acetic acid while stirring.

[0298] The XylX6 Stock Substrate was prepared by addition of 5mL of Milli-Q water to 1 bottle of XylX6 substrate. The solution was mixed well until thoroughly dissolved. Aliquot 0.75mL of substrate stock into 4mL amber vials and stored at -20°C until use. The XylX6 working Substrate was prepared by 1 vial of frozen stock substrate, 0.75mL, was added 3mL of 12 plato all-malt wort pH 5.6 (as described above) and mixed thoroughly. Mcllvaine Buffer pH 5.0 (Assay Buffer) was prepared by dissolving 10.19g of citric acid (monohydrate) and 18.33g of disodium hydrogen phosphate dihydrate into 900 mL of Milli-Q water. pH was adjusted if needed by IM HC1 or ImNaOH. 2% Tris Stop Solution was prepared by dissolving 20g of Trizma base into 900 mL of Milli-Q water. No pH adjustment was needed.

[0299] The following xylanase samples; Xylanase variant from Aspergillus kawachii (AkaXyl_var) having the mature amino acid sequence shown in SEQ ID NO:9. A preparation of BsbBglu + AkaXyl_var (having a xylanase activity of 13680 - 16720 XBU / g and having an 0- glucanase activity of 9090 - 12500 BBU / g) and FveXyn4_l as set in SEQ IS NO 1 were both obtained from Dupont Nutrition Bioscience Aps, Denmark, Shearzyme P105 and Ultraflo Max from Novozymes, Denmark. All xylanase samples were diluted 1g in 9g 12°Plato wort pH 5.6. To test thermostability of the xylanase in wort, ImL of the diluted xylanase samples was in Eppendorf tubes incubated ranging from 20 to 90°C for 10 minutes using an Eppendorf Thermomixer C (Eppendorf, Hamburg) and followingly immediately put on ice before determination of the residual xylanase activity.

[0300] Xylanase activity was determined in MTP format with liquid handling using Biomek robots (Beckman Coulter, USA). lOOpI of the freshly prepared reaction substrate XylX6 was transferred (using Sodium Acetate buffer pH 5.0, working substrate) to the microtiter plate. 15pl of the enzyme sample appropriately diluted in Mcllvaine Buffer pH 5.0 was added to ensure Abs405nm between 0.0- 1.0 for all temperatures. The same enzyme dilution is used across various temperature tests. The enzyme is mixed with substrate and plate is sealed with tape. Incubation of each plate for 1800 sec at 30°C with 200 rpm shaking is applied and reaction is stopped using lOOpL 2% Tris Stop Solution. The absorbance was measured at 405 nm in a MTP-reader (Molecular Devices Spectramax 190) was determined for reactions. Blank reaction is constructed using Mcllvaine Buffer pH 5.0 instead of enzyme and Abs405nm for each sample is subtracted Abs405nm for blank. All reactions are determined as average of duplicates. The relative activity (Abs405nm) of a given temperature is plotted against the highest Abs405nm obtained for the given temperatures.

[0301] The results of the thermostability test in the 100% malt-based wort pH 5.6 is shown in figure 7. It is clear that FveXyn4_l show significant higher thermostability than the tested xylanase samples known for mashing use (AkaXyl_var, Shearzyme Pl 05 and Ultraflo Max). FveXyn4_l showed 100% residual activity at 70°C and 63% residual activity at 80°C indicative of optimal malt based mashing extending from 50°C to 78°C.

[0302] Example 16 - Xylanase stability during malt-based mashing

[0303] Xylanase stability was tested in various high temperature mashing programs

[0304] Various 40% barley + 60% malt mashing programs was prepared using water-to-grist ratio of 3:1. Enzymes was tested in a mashing operation model system for wort production using milled Malt (Pilsner Malt from Sophus Fuglsang, Denmark, Batch Number 14.12.2022) at a Buhler Miag malt mill 0.5 mm setting, position 4) and milled Barley (Fuglsang, Denmark, Batch Number 14.12.2022) milled at a Buhler Miag malt mill 0.5 mm setting, position 4).

[0305] As an example of XIP un-inhibited xylanase, FveXyn4_l was applied and as an example of a XIP inhibited xylanase AkaXyl_var, Shearzyme P105 and Ultraflo Max were applied. Xylanase variant from Aspergillus kawachii (AkaXyl_var) having the mature amino acid sequence shown in SEQ ID NO:9. A preparation of BsbBglu + AkaXyLvar (having a xylanase activity of 13680 - 16720 XBU / g and having an |3-glucanase activity of 9090 - 12500 BBU / g) and FveXyn4_l as set in SEQ IS NO 1 were both obtained from Dupont Nutrition Bioscience Aps, Denmark and Ultraflo Max from Novozymes, Denmark

[0306] Mashing operation for wort production

[0307] Barley grist (28.0g milled Barley grains) and Malt grist (42.0g milled Malt grains) were mixed in beakers with 210g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid, resulting in a water to grist ratio of 3.0:1. The FveXyn4_l xylanase variant was added based on mg protein determined according to example 4 and dosed at 68.0 pg FveXyn4_l protein per g Grist for comparison. AkaXyl_var was dosed 4,3 XBU per g Grist and 0.3mg Ultraflo Max per g Grist. The grist was mashed with three mashing programs (1-3) listed in table 18 to 20;

[0308] Table 18, Main mashing programs low temperature mashing

[0309] Table 19, Main mashing programs standard temperature mashing

[0310]

[0311] Table 20, Main mashing programs high temperature mashing

[0312] Mash samples was taking out (5mL) from Main mash at each individual temperature indicated in table 18 to 20 and immediately cooled to 5°C, followingly centrifuged 12000 rpm in tabletop centrifuge, and supernatant of mash sample stored at -20°C for later xylanase activity analysis.

[0313] Residual xylanase activity was determined in mash samples using XylX6 substrate as described in example 15. Blank reaction is constructed using tap water instead of enzyme addition to the mash and Abs405nm of blank for each sample was subtracted Abs405nm for each mash samples. Xylanase activity of all mash samples were determined as average of duplicates. The relative activity (Abs405nm) of a given mash sample was set relative to the activity of xylanase activity of the initial activity (0 min) and the relative xylanase activity was plotted against sampling during the various mashing programs.

[0314] The residual xylanase activity during mashing programs 1 , 2 and 3 are shown in figure 8 A, B and C respectively. It can be seen, that FveXyn4_l has significant higher residual xylanase through various mashing profiles, maintaining at least 75% xylanase activity at end of mash off (78°C) compared to no determined activity of AkaXyl_var, and Ultraflo Max (0%) at mash off. This may enable better mash filtration by xylanase activity in end mashing to reduce viscosity. Example 17 - Influence of cereal extracts on Beta-glucanase activity

[0315] A limiting factor for overall mash filtration performance is both efficiency of xylanase and Beta-glucanase action on various cereals that may be the present. Thus, beside the xylanase inhibition by raw material inhibitors we tested beta-glucanase performance in presence of milled grain used in mashing for beer production. Proteinaceous soluble inhibitors of xylanases been found in barley, rye, com and other cereals. The example describes a simple and novel assay for Beta-glucanase inhibition / action by presence wheat, corn, rye and malt. The inhibition degree of wheat, com, rye and malt was determined for various Beta-glucanase products.

[0316] Cereal extracts were prepared by the following procedure. A weighted amount of either rye, or wheat flour (Valsempllen, Denmark) (20 g) or fine milled com grist or Malt (Buhler Miag malt mill 0.5 mm setting, position 4) (20g) was suspended in 200 mL (10% w / v, DS) of 0.1 M Na-acetate buffer, pH 5.6, and the mixture agitated on a shaker at 300 rpm for 3.0 hr. The insoluble portion was separated by centrifugation at 5,000 rpm for 20 min in a swing-bucket centrifuge. The supernatants were treated as Rye, Wheat, Corn and Malt extracts pH 5.6 respectively. Dilution series ranging from 500 - 1000000 times, were prepared using 0.1 M Na- acetate buffer, pH 5.6, with the following Beta-glucanase samples; BsbBglu + FveXyn4_l (having a xylanase activity of 15969 - 19517 NGXU / g and having a Beta-glucanase activity of 22356 - 27324 BBXU / g) obtained from Dupont Nutrition Bioscience Aps, Denmark, Ultraflo Max and Filtrase Fast from Novozymes, Denmark and DSM, respectively. All combinations of diluted enzyme samples were incubated 24 hours at 5 °C to facilitate potential inhibition and following 1 mL 0.1 M Na-acetate buffer, pH 5.6 was mixed with 1 mL of the diluted enzyme sample in a reaction tube and placed 5 min at 40°C using a waterbath for equilibration. One Beta-glucazyme tab purchased from Megazyme (Beta-glucazyme, T-BGZ 12 / 12 to assay endo- beta-Glucanase activity on Azurine-crosslinked barley P-glucan from Megazyme, Ireland) was added to each reaction tube, incubated 10 minutes at 40°C and all reactions were stopped by addition of 10 mL demineralized water according to manufactures declaration. The reaction solution was filtered by paper filter (Whatman, Cytiva Grade 597 Plus Qualitative Filter Paper, Circles) and absorbance at 590nm was determined using a Spectrophotometer (Genesys Spectrophotometer 10S UV-Vis). The relative Beta-glucanase activity was calculated as % compared to the buffer without any cereal extracts (Rye, Wheat, Com, and Malt) only using the enzyme sample dilution resulting in absorbance values ranging between 0.0 and 1.0 in assay.

[0317] The result of testing beta-glucanase activity in samples using cereal extracts (Rye, Wheat, Corn, and Malt) are shown in figure 9. It may be seen that BsbBglu + FveXyn4_l demonstrated higher residual activity (360%) with malted barley than Ultraflo Max (271%) and Filtrase Fast (306%). All residual activities of BsbBglu + FveXyn4_l was notably higher as compared to no cereal addition as comped to the other tested beta-glucanases. Thus, BsbBglu do not seem inhibited by the tested cereal extracts (malt barley, corn, wheat and rye) as seen for the other beta-glucanase samples.

[0318] Example 18 - Combination of beta-glucanase and xylanase performance in Malt mashing trials with Barley inclusion

[0319] The objective of this example was to test a Rye XIP un-inhibited vs inhibited xylanase in combination with un-inhibited Beta-glucanase present during a 60% malt: 40% barley based mashing process, using various xylanase dosages. Enzymes was tested in a mashing operation model system for wort production using milled Malt (Pilsner Malt from Sophus Fuglsang, Denmark, Batch Number 10.12.2019) at a Buhler Miag malt mill 0.5 mm setting, position 4, milled barley (Raw Barley from Sophus Fuglsang, Denmark, Batch Number 09.03.2019) at a Buhler Miag malt mill 0.5 mm setting, position 4. As an example of a Rye XIP un-inhibited xylanase, FveXyn4_l (17.9% inhibition) was applied and as an example of a Rye XIP inhibited xylanase was AkaXyl_var (99.9% inhibition) applied as well as, according to Rye XIP inhibition as described in example 9. The combination of BsbBglu + FveXyn4_l (having a xylanase activity of 15969 - 19517 NGXU / g and having a Beta-glucanase activity of 22356 - 27324 BBXU / g) as well as BsbBglu + AkaXyl_var (having a xylanase activity of 13680 - 16720 XBU / g and having an [3-glucanase activity of 9090 - 12500 BBU / g) were obtained from Dupont Nutrition Bioscience Aps and Ultraflo Max from Novozymes, Denmark

[0320] Mashing operation for wort production

[0321] Barley grist (28.0g milled Barley grains) and Malt grist (42.0g milled Malt grains) were mixed in beakers with 210g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid, resulting in a water to grist ratio of 3.1 : 1 . The enzyme materials were all added based on g per kg grist for comparison (0.025, 0.05 and O.lg / kg).

[0322] The grist was mashed with the program; heated to 60°C for mashing in and pH adjustment before enzymes were applied and hereafter kept at 60°C for 5 minutes; heated to 65°C for 5 minutes by increasing temperature with l°C / minute; kept at 65°C for 45 minutes; heated to 72°C for 7 minutes by increasing temperature with l°C / minute; and kept at 72°C for another 20 minutes; heated to 78°C for 6 minutes by increasing temperature with l°C / minute; and kept at 78°C for another 10 minutes - mashing off; content of each sample was adjusted to 350g and filtered. The volumes of the wort collected after 5, 10, 15 and 30 minutes were measured, and pH was adjusted to pH 5.2 with 2.5 M sulphuric acid. One pellet of hops was added to each flask. The wort samples were boiled for 60 minutes in a boiling bath and samples were cooled down to 17°C. All the samples were filtered used for analysis, see below.

[0323] Wort sample viscosity were determined as described in example 6 and the results shown for all dosages (0.025, 0.05 and O.lg / kg) for all Beta-glucanase and xylanase samples. The wort sample viscosity is shown in Figure 10 A.

[0324] The content of High Molecular Weight (HMW) B-glucan was measured in the wort using a Microtitre plate reader from BMG Labtech (FLUOstar Omega) following Standard Instruction Brewing, 23.8580-B30, Based on Microtitre method, from EBC 8.13.2. Wort sample B-glucan content is shown in Figure 10 B).

[0325] The content of High Molecular Weight (HMW) pentosan was measured in wort samples following Standard Instruction Brewing, 23.8580-B39. (Based on Advanced, A3605 precipitation with 96 (w / w)% ethanol followed by acid hydrolysis). Wort sample HMW pentosan content is shown in Figure 10 C).

[0326] It can be observed that the uninhibited xylanase and Beta-glucanase (BsbBglu + FveXyn4_l) showed decreased wort viscosity with low amount of HMW b-glucan and pentosan as compared to the inhibited blends of beta-glucanase and xylanase (BsbBglu + AkaXyl_var) and Ultraflo Max. It may be preferable to use an un-inhibited xylanase and un-inhibited Beta- glucanase such as BsbBglu + FveXyn4_l to obtain low wort viscosity and very efficient mash performance of malt with other cereals such as Barley, Rye, Wheat and Corn.

Claims

CLAIMSWhat is claimed is:

1. A method for producing a rye whiskey comprising the steps of:(a) providing a grist comprising 51 w / w % of rye grain;(b) adding water to the grist 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 inhibitor;(f) saccharifying the mash of step (e);(g) fermenting the mash of step (f) to produce a fermentate containing ethanol;(h) distilling the fermentate of step (g) to provide the rye whiskey.

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

3. The method of claim 2 wherein the xylanase has less than 20% inhibition by rye XIP inhibitor.

4. The method of any preceding claim 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 xylanasc is an enzyme having xylanasc 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 of the preceding claims wherein the rye grain is malted.

15. The method of any of claims 1 to 13 wherein the rye grain is unmalted.

16. The method of any of claims 1 to 15 wherein the rye grain is milled.

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

18. The method of any of the preceding claim wherein the grist further comprises malted or unmalted barley, wheat, com, rye, rice, cassava, oatmeal or sorghum.

19. The method of claim 18 wherein the grist further comprises malted barley.

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

21. The method of any of the preceding claims further comprising adding an alpha-amylase and / or a beta-glucanase at any 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 active fragment thereof.

26. The method of claim 25 wherein the beta-glucan ase is an enzyme having beta-glucan ase activity comprising a polypeptide having at least 90% sequence identity to SEQ ID NO:8 or a beta-glucanase active 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 active 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 active 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 active 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 active 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 active 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 at step (f .

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

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

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

37. The method of claim 36 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 NOTO or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NOT 1 or an alpha-L-arabinofuranosidase active fragment thereof.

38. The method of claim 37 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 NOTO or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO:11 or an alpha-L-arabinofuranosidase active fragment thereof.

39. The method of claim 38 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 NOTO or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO:11 or an alpha-L-arabinofuranosidase active fragment thereof.

40. The method of claim 39 wherein the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity comprising a polypeptide having at least 92% sequence identity to SEQ ID NOTO or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NOT 1 or an alpha-L-arabinofuranosidase active fragment thereof.

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

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

43. The method of claim 42 wherein the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NOTO or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NOT 1 or an alpha-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 NOTO 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 a feruloyl esterase at any of steps (b), (c), (d) and / or (e).

46. The method of claim 45 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.

47. The method of claim 46 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.

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

49. The method of claim 48 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.

50. The method of claim 49 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.

51. The method of claim 50 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.

52. The method of claim 51 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.

53. The method of claim 52 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.

54. The method of claim 53 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.

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

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

57. The method of claim 56 wherein the xylanase has less than 20% inhibition by Rye XIP inhibitor.

58. The method of any of claims 55 to 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 uninhibited 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 of claims 55 to 67 wherein the rye grain is malted.

69. The method of any of claims 55 to 67 wherein the rye grain is unmalted.

70. The method of any of claims 55 to 69 wherein the rye grain is milled.

71. The method of any of claims 55 to 70 wherein the grist 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 of claims 55 to 71 wherein the grist further comprises malted or unmalted barley, corn, rye, wheat, cassava, oatmeal or sorghum.

73. The method of claim 72 wherein the grist further comprises malted barley.

74. The method of any 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 active 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 active fragment thereof.

83. The method of claim 82 wherein the beta-glucan ase is an enzyme having beta-glucan ase activity comprising a polypeptide having at least 99% sequence identity to SEQ ID NO:8 or a beta-glucanase active 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 active 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 of claims 55 to 85 further comprising adding an alpha-L- arabinofuranosidase at steps (a) and / or (b).

87. The method of claim 85 wherein the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofurano sidase activity comprising a polypeptide having at least 70% sequence identity to SEQ ID NOTO or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active 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 NOTO or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO:11 or an alpha-L-arabinofuranosidase active 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 NOTO or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NOT 1 or an alpha-L-arabinofuranosidase active 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% sequenceidentity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha- L-arabinofuranosidasc active fragment thereof.

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

92. The method of claim 91 wherein the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity comprising a polypeptide having at least 95% sequence identity to SEQ ID NOTO or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NOT 1 or an alpha-L-arabinofuranosidase active fragment thereof.

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

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

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

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

97. The method of claim 96 wherein the feruloyl esterase is an enzyme having femloyl 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 fcruloyl esterase is an enzyme having fcruloyl 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 that has reduced sensitivity to a cereal XIP inhibitor comprising assaying the activity of the xylanase in the presence and absence of the cereal XIP inhibitor and determining the percent inhibition by the cereal XIP inhibitor.

107. The method of claim 106 wherein the cereal is barley, wheat, corn, rye, rice, cassava, oatmeal 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. Th method of claim 108 wherein the cereal is wheat.

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

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

113. The xylanase of claim 112 having less than 20% inhibition by Rye XIP inhibitor.

114. The xylanase of claim 113 comprising 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 1 14 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 xylanasc of claim 122 comprising an enzyme having xylanasc activity comprising a polypeptide according to SEQ ID NO:2 or SEQ ID NO:3.

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

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

126. The xylanase of claim 125 having less than 55% inhibition by wheat XIP inhibitor.

127. The xylanase of claim 126 comprising 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 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.

129. The xylanase of claim 128 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.

130. The xylanase of claim 129 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.

131. The xylanase of claim 130 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.

132. The xylanasc of claim 131 comprising an enzyme having xylanasc 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 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.

134. The xylanase of claim 133 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.

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 comprising a xylanase having reduced sensitivity to wheat XIP inhibitor and a beta-glucanase.

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 xylanaseactive fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the bcta-glucanasc is an enzyme having bcta-glucanasc 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 thebeta-glucanase is an enzyme having beta-glucanase activity comprising a polypeptide having at least 98% sequence identity to SEQ ID NO:8 or a bcta-glucanasc 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 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 preparing a low viscosity mash comprising the steps of:(a) preparing a mash from a grist comprising wheat grain in the presence of a composition according to any of claims 137 to 147;(b) optionally filtering the mash to obtain a wort.

149. The method of claim 148 wherein the wheat grain is malted.

150. The method of claim 148 wherein the wheat grain is unmalted.

151. The method of any of claims 148 to 150 wherein the wheat grain is milled.

152. The method of any of claims 148 to 151 wherein the grist 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 of claims 148 to 152 wherein the grist further comprises malted or unmalted barley, corn, rye, rice, cassava, oatmeal or sorghum.

154. The method of claim 153 wherein the grist further comprises malted barley.

155. A composition comprising a xylanase comprising a polypeptide having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof and a glucanase.

156. The composition of claim 155 wherein said xylanase polypeptide has at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.

157. The composition of claim 156 wherein said xylanase polypeptide has at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.

158. The composition of claim 157 wherein said xylanase polypeptide has at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.

159. The composition of claim 158 wherein said xylanase polypeptide has at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.

160. The composition of claim 159 wherein said xylanase polypeptide has at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.

161. The composition of claim 160 wherein said xylanase polypeptide has at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.

162. The composition of claim 161 wherein said xylanase polypeptide comprises a sequence according to SEQ ID NO:2 or a xylanase active fragment thereof.

163. The composition of claim 162 wherein said xylanasc polypeptide comprises a sequence according to SEQ ID NO:2.

164. The composition of any of claims 155 to 163 wherein said glucanase comprises a polypeptide having at least 80% sequence identity to SEQ ID NO: 8 or a glucanase active fragment thereof.

165. The composition of claim 164 wherein said glucanase polypeptide has at least 85% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.

166. The composition of claim 165 wherein said glucanase polypeptide has at least 90% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.

167. The composition of claim 166 wherein said glucanase polypeptide has at least 92% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.

168. The composition of claim 167 wherein said glucanase polypeptide has at least 95% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.

169. The composition of claim 168 wherein said glucanase polypeptide has at least 98% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.

170. The composition of claim 169 wherein said glucanase polypeptide has at least 99% sequence identity to SEQ ID NO: 8 or a glucanase active fragment thereof.

171. The composition of claim 170 wherein said glucanase polypeptide comprises a sequence according to SEQ ID NO: 8 or a glucanase active fragment thereof.

172. The composition of claim 171 wherein said glucanase polypeptide comprises a sequence according to SEQ ID NO:8.

173. The composition of any of claims 164 to 172 wherein the xylanasc polypeptide has at least 98% sequence identity to SEQ ID NO:2.

174. The composition of claim 173 where in the xylanase polypeptide has at least 99% sequence identity to SEQ ID NO:2.

175. The composition of claim 174 wherein the xylanase polypeptide comprises a sequence according to SEQ ID NO:2.

176. The composition of claim 175 wherein said glucanase polypeptide comprises a sequence according to SEQ ID NO:8.

177. The composition of any of claims 155 to 176 wherein the composition is a liquid.

178. The composition of claim 177 wherein the xylanase is present in an amount of 1,000 to 50,000 NGXU / g and the glucanase is present in an amount of 1,000 to 50,000 BBXU / g.

179. The composition of claim 178 wherein the xylanase is present in an amount of 5,000 to 30,000 NGXU / g and the glucanase is present in an amount of 5,000 to 30,000 BBXU / g.

180. The composition of claim 179 wherein the xylanase is present in an amount of 10,000 to 25,000 NGXU / g and the glucanase is present in an amount of 15,000 to 30,000 BBXU / g.

181. The composition of claim 180 wherein the wherein the xylanase is present in an amount of 15,000 to 20,000 NGXU / g and the glucanase is present in an amount of 20,000 to 28,000 BBXU / g.

182. A method of altering filterability of a starch comprising material, said method comprising the step of treating said starch comprising material with a composition according to any of claims 137 to 147 and 155 to 181.

183. A method of reducing pressure built up during lautcring in a brewing application, said method comprising the step of treating a brewing mash comprising a starch containing material with a composition according to any of claims 137 to 147 and 155 to 181.

184. A method for the production of a food, feed, or beverage product, such as an alcoholic or non-alcoholic beverage, such as a cereal- or malt-based beverage like beer or whiskey, said method comprising the step of treating a starch comprising material with a composition according to any of claims 137 to 147 and 155 to 181.185 A method for the production of a brewing mash, said method comprising the step of treating a starch comprising material with a composition according to any of claims 137 to 147 and 155 to 181.

186. The method of any of claims 182 to 185 wherein the starch comprising material comprises a grist.

187. The method of claim 186 wherein the grist comprises at least 50% malt.

188. The method of claim 187 wherein the grist comprises at least 60% malt.

189. The method of claim 188 wherein the grist comprises at least 70% malt.

190. The method of claim 189 wherein the grist comprises at least 80% malt.

191. The method of claim 189 wherein the grist comprises at least 90% malt.

192. The method of any of claims 187 to 191 wherein the grist further comprises wheat, corn or rye.

193. The method of any of claims 186 to 192 wherein 0.01 to 1 grams of the composition is added per Kg of grist.

194. The method of claim 193 wherein 0.02 to 0.1 grams of the composition is added per Kg of grist.

195. A method for preparing a low viscosity mash comprising the steps of:(a) preparing a mash from a grist in the presence of a xylanase having reduced sensitivity to rye XIP inhibitor and a beta-glucanase which has increased activity in the presence of at least 10% rye compared to a sample not having rye;(b) optionally filtering the mash to obtain a wort.

196. The method of claim 195 wherein the xylanase comprises a polypeptide having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.

197. The method of claim 196 wherein the xylanase comprises a polypeptide having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.

198. The method of claim 197 wherein the xylanase comprises a polypeptide having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.

199. The method of claim 198 wherein the xylanase comprises a polypeptide having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.

200. The method of claim 199 wherein the xylanase comprises a polypeptide having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.

201. The method of claim 200 wherein the xylanase comprises a polypeptide having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.

202. The method of claim 201 wherein the xylanase comprises a polypeptide having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.

203. The method of claim 202 wherein the xylanase comprises a polypeptide according to SEQ ID NO:2 or a xylanase active fragment thereof.

204. The method of claim 203 wherein the xylanase comprises a polypeptide according to SEQ ID NO:2.

205. The method of any of claims 195 to 204 wherein the glucanase comprises a polypeptide having at least 80% sequence identity to SEQ ID NO: 8 or a glucanase active fragment thereof.

206. The method of claim 205 wherein the glucanase comprises a polypeptide having at least 85% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.

207. The method of claim 206 wherein the glucanase comprises a polypeptide having at least 90% sequence identity to SEQ ID NO: 8 or a glucanase active fragment thereof.

208. The method of claim 207 wherein the glucanase comprises a polypeptide having at least 92% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.

209. The method of claim 208 wherein the glucanase comprises a polypeptide having at least 95% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.

210. The method of claim 209 wherein the glucanase comprises a polypeptide having at least 98% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.

211. The method of claim 210 wherein the glucanase comprises a polypeptide having at least 99% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.

212. The method of claim 21 1 wherein the glucanase comprises a polypeptide according to SEQ ID NO:8 or a glucanase active fragment thereof.

213. The method of claim 212 wherein the glucanase comprises a polypeptide according to SEQ ID NO:8.

214. The method of any of claims 195 to 213 wherein the grist comprises at least 50% malt.

215. The method of claim 214 wherein the grist comprises at least 60% malt.

216. The method of claim 215 wherein the grist comprises at least 70% malt.

217. The method of claim 216 wherein the grist comprises at least 80% malt.

218. The method of claim 217 wherein the grist comprises at least 90% malt.

219. The method of any of claims 214 to 218 wherein the grist further comprises wheat, corn or rye.

220. The method of claim 219 wherein the grist further comprises rye.

221. The method of claim 220 wherein the grist comprises at least 10% rye (w / w).

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