TECHNICAL FIELD
[001] The subject matter described herein in general relates to food products, and in particular, relates to enzymatic compositions for preparing reduced gluten food products.
BACKGROUND OF INVENTION
[002] The number of individuals suffering from gluten sensitivity or gluten intolerance is increasing by folds in these days. Gluten containing diet when ingested by these individuals would cause adverse immunological reactions ultimately leading to enteropathy and malabsorption syndrome. (Shewry PR., Wheat; J Exp Bot. 2009;60(6): 1537-53). To cater to the requirements of patients suffering from gluten-related disorders, the primary health care providers mainly ask the patients to adhere to a gluten-free diet or very-low gluten diet. However, because of its presence in several cereal grains such as barley, wheat, rye, and food products like pasta, malt, semolina, oats, energy bars, etc., it becomes extremely difficult to restrict or prohibit gluten containing food.
[003] Attempts to make gluten free food products meant a compromise on texture, viscoelastic behavior and cohesiveness to the food products. For example, a dough prepared by mixing gluten free cereal flour does not rise as efficiently as the gluten containing flour. In certain food products, water soluble cellulose ether and isabgol were used to render the bread voluminous, as disclosed in Carbohydr. Poly., 25, 337 (1994). It is also known to add water-soluble polysaccharides such as guar gum and xanthan gum for the same purpose. However, the bread made using isabgol or water-soluble polysaccharides has a certain volume, but a hard crust, giving a disrelish to the mouth. When water-soluble polysaccharides are added, the crumb is elastic enough to give a rubber-like mouthfeel. When food fibres like isabgol are added, the crumb is very tender and non-elastic, giving a mouthfeel far different than bread. Due to poor moisture retention, the bread becomes dry and crumbly with the lapse of time from baking, suddenly losing a mouthfeel (EP1561380B1). Although various methods to produce hypoallergic or reduced-gluten wheat flour are known in the art, these methods are time

consuming, cost ineffective, temperature and pH specific involving multimodal
steps.
SUMMARY OF THE INVENTION
[004] In an aspect of the present invention, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1. [005] In an aspect of the present invention, there is provided a process for preparing an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1, said process comprising steps of: a) obtaining the protease enzyme (EC 3.4.23.18); b) obtaining the xylanase enzyme (EC 3.2.1.8); and c) contacting the protease enzyme (EC 3.4.23.18) with the xylanase enzyme (EC 3.2.1.8) to obtain the composition.
[006] A gluten-reduced dough obtained using the enzymatic composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1.
[007] A baked product obtained using the enzymatic composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1.
[008] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF THE DRAWINGS

[009] The following drawings form part of the present specification and are
included to further illustrate aspects of the present disclosure. The disclosure may
be better be understood by reference to the drawings in combination with the
detailed description of the specific embodiments presented herein.
[0010] Figure 1 represents the effect of various enzymes at a combined
concentration of lOOppm with respect to percentage reduction in gluten in
comparison to the control, in accordance with an embodiment of present disclosure.
[0011] Figure 2 represents the effect of protease at various concentrations towards
reduction of gluten in wheat flour, in accordance with an embodiment of present
disclosure.
[0012] Figure 3 represents effect of xylanase at various concentrations towards
reduction of gluten in wheat flour, in accordance with an embodiment of present
disclosure.
[0013] Figure 4 represents effect of combination of protease and xylanase at
various w/w ratios, in accordance with an embodiment of present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Those skilled in the art will be aware that the present disclosure is subject to
variations and modifications other than those specifically described. It is to be
understood that the present disclosure includes all such variations and
modifications. The disclosure also includes all such steps, features, compositions,
and compounds referred to or indicated in this specification, individually or
collectively, and any and all combinations of any or more of such steps or features.
Definitions
[0015] For convenience, before further description of the present disclosure,
certain terms employed in the specification, and examples are delineated here.
These definitions should be read in the light of the remainder of the disclosure and
understood as by a person of skill in the art. The terms used herein have the
meanings recognized and known to those of skill in the art, however, for
convenience and completeness, particular terms and their meanings are set forth
below.

[0016] The articles "a", "an" and "the" are used to refer to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
[0017] The terms "comprise" and "comprising" are used in the inclusive, open
sense, meaning that additional elements may be included. It is not intended to be
construed as "consists of only".
[0018] Throughout this specification, unless the context requires otherwise the
word "comprise", and variations such as "comprises" and "comprising", will be
understood to imply the inclusion of a stated element or step or group of element or
steps but not the exclusion of any other element or step or group of element or
steps.
[0019] The term "including" is used to mean "including but not limited to".
"Including" and "including but not limited to" are used interchangeably.
[0020] Ratios, concentrations, amounts, and other numerical data may be presented
herein in a range format. It is to be understood that such range format is used
merely for convenience and brevity and should be interpreted flexibly to include
not only the numerical values explicitly recited as the limits of the range, but also
to include all the individual numerical values or sub-ranges encompassed within
that range as if each numerical value and sub-range is explicitly recited. For
example, a temperature ranges of about 25-35 °C should be interpreted to include
not only the explicitly recited limits of about 25 °C to about 35 °C, but also to
include sub-ranges, such as 25-30 °C, 28-35 °C, and so forth, as well as individual
amounts, including fractional amounts, within the specified ranges, such as 25.2 °C,
and 32.5 °C, for example.
[0021] The term "at least one" is used to mean one or more and thus includes
individual components as well as mixtures/combinations.
[0022] The term "composition" and "enzymatic composition" have been used
interchangeably throughout the specification.
[0023] Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to
which this disclosure belongs. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or testing of the

disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference. [0024] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein. [0025] As mentioned previously, conventionally methods to reduce gluten are based on avoiding or restricting foods that contain gluten. However, given the vast majority of foods that contain gluten, abstaining from such food products is a quite challenging. Also, currently used gluten substitutes are a compromise on texture, taste and/or consistency. Conventionally used methods to make reduced-gluten wheat flour are time consuming, cost ineffective, temperature and pH specific involving multimodal steps. The present invention is directed towards the use of an enzymatic composition to reduce gluten in dough of various cereal grains and food products. Although the description herein presented refers to reduced gluten in wheat dough, the proposed composition may also be used to reduce gluten in other cereal grains such as rye, wheat, barley, and food products, including, but not limited to, pasta, energy bars, noodles, breads and pastries, baked foods such as cakes, cookies, pie crusts, brownies, tortillas, beer and malt beverages. [0026] In an embodiment of the present invention, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1. In another embodiment the enzyme composition comprises a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4.33:1 to 15:1. In yet another embodiment the enzyme composition comprises a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 5:1 to 16:1.

[0027] In an embodiment of the present disclosure, there is provided an enzyme
composition as disclosed herein, wherein the protease enzyme (EC 3.4.23.18) to
the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4.33:1 to 15:1.
[0028] In an embodiment of the present disclosure, there is provided an enzyme
composition comprising a) a protease enzyme (EC 3.4.23.18); and b) a xylanase
enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase
enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4.33:1 tol5:l.
[0029] In an embodiment of the present disclosure, there is provided an enzyme
composition as disclosed herein, wherein the protease enzyme (EC 3.4.23.18) to
the xylanase enzyme (EC 3.2.1.8) has a w/w ratio of 4.33:1.
[0030] In an embodiment of the present disclosure, there is provided an enzyme
composition as disclosed herein, wherein the protease enzyme (EC 3.4.23.18) to
the xylanase enzyme (EC 3.2.1.8) has a w/w ratio of 6.4:1.
[0031] In an embodiment of the present disclosure, there is provided an enzyme
composition comprising a) a protease enzyme (EC 3.4.23.18); and b) a xylanase
enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase
enzyme (EC 3.2.1.8) has a w/w ratio is 4.33:1.
[0032] In an embodiment of the present disclosure, there is provided an enzyme
composition comprising a) a protease enzyme (EC 3.4.23.18); and b) a xylanase
enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase
enzyme (EC 3.2.1.8) has a w/w ratio is 6.4:1.
[0033] In an embodiment of the present disclosure, there is provided an enzyme
composition as disclosed herein, wherein the protease enzyme (EC 3.4.23.18) to
the xylanase enzyme (EC 3.2.1.8) has a w/w ratio of 7:1.
[0034] In an embodiment of the present disclosure, there is provided an enzyme
composition comprising a) a protease enzyme (EC 3.4.23.18); and b) a xylanase
enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase
enzyme (EC 3.2.1.8) has a w/w ratio is 7:1.
[0035] In an embodiment of the present disclosure, there is provided an enzyme
composition as disclosed herein, wherein the protease enzyme (EC 3.4.23.18) to
the xylanase enzyme (EC 3.2.1.8) has a w/w ratio of 15:1.

[0036] In an embodiment of the present disclosure, there is provided an enzyme composition comprising a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio is 15:1.
[0037] In an embodiment of the present disclosure, there is provided an enzyme composition as disclosed herein, wherein the protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600 XU/g. In another embodiment, the protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 9,000-11,000 PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3800-4500 XU/g. In yet another embodiment, the protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 10,000 PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 4000 XU/g. [0038] In an embodiment of the present disclosure, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1, and wherein the protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600 XU/g.
[0039] In an embodiment of the present disclosure, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio is 4.33:1, and wherein the protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600 XU/g.
[0040] In an embodiment of the present disclosure, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio is 6.4:1, and wherein the protease enzyme (EC

3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the
xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600
XU/g.
[0041] In an embodiment of the present disclosure, there is provided an enzyme
composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase
enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase
enzyme (EC 3.2.1.8) has a w/w ratio is 7:1, and wherein the protease enzyme (EC
3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the
xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600
XU/g.
[0042] In an embodiment of the present disclosure, there is provided an enzyme
composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase
enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase
enzyme (EC 3.2.1.8) has a w/w ratio is 15:1, and wherein the protease enzyme (EC
3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the
xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600
XU/g.
[0043] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition further comprises water.
[0044] In an embodiment of the present disclosure, there is provided an enzyme
composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase
enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase
enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1, and wherein the
composition further comprises water.
[0045] In an embodiment of the present disclosure, there is provided an enzyme
composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase
enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase
enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1, and wherein the
protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 8500-11500
PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of
3400-4600 XU/g, and wherein the composition further comprises water.

[0046] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition. In another embodiment, the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-94 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 7-13 ppm with respect to the composition.
[0047] In an embodiment of the present disclosure, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition.
[0048] In an embodiment of the present disclosure, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4.33:1 to 15:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition.
[0049] In an embodiment of the present disclosure, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio is 4.33:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition.
[0050] In an embodiment of the present disclosure, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase

enzyme (EC 3.2.1.8) has a w/w ratio is 6.4:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition.
[0051] In an embodiment of the present disclosure, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio is 7:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition.
[0052] In an embodiment of the present disclosure, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio is 15:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition.
[0053] In an embodiment of the present disclosure, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600 XU/g, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition. [0054] In an embodiment of the present disclosure, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); b) a xylanase enzyme (EC 3.2.1.8), and c) water, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and

wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition.
[0055] In an embodiment of the present disclosure, there is provided an enzyme composition comprising: a) a protease enzyme (EC 3.4.23.18); b) a xylanase enzyme (EC 3.2.1.8), and c) water, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600 XU/g, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition. [0056] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the composition further comprises at least one excipient. In an embodiment, the at least one excipient is food product or a cereal grain flour. In another embodiment, the at least one excipient is wheat flour. In yet another embodiment the at least one excipient is a pH regulator. [0057] In an embodiment of the present disclosure, there is provided an enzymatic composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the composition comprises at least one excipient.
[0058] In an embodiment of the present disclosure, there is provided an enzymatic composition comprising: a) a protease enzyme (EC 3.4.23.18); b) a xylanase enzyme (EC 3.2.1.8), c) water and d) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1.
[0059] In an embodiment of the present disclosure, there is provided an enzymatic composition comprising: a) a protease enzyme (EC 3.4.23.18); b) a xylanase enzyme (EC 3.2.1.8), c) water and d) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the

range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600 XU/g. [0060] In an embodiment of the present disclosure, there is provided an enzymatic composition comprising: a) a protease enzyme (EC 3.4.23.18); b) a xylanase enzyme (EC 3.2.1.8), c) water and d) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition. [0061] In an embodiment of the present disclosure, there is provided an enzymatic composition comprising: a) a protease enzyme (EC 3.4.23.18); b) a xylanase enzyme (EC 3.2.1.8), c) water and d) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600 XU/g, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition.
[0062] In an embodiment of the present disclosure, there is provided an enzymatic composition comprising: a) a protease enzyme (EC 3.4.23.18); b) a xylanase enzyme (EC 3.2.1.8), c) water and d) wheat flour, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4.33:1 - 15:1, and wherein the protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600 XU/g, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition.

[0063] A gluten-reduced dough obtained using the enzymatic composition
comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC
3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC
3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1.
[0064] A gluten-reduced dough obtained using the enzymatic composition
comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC
3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC
3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1, and wherein the protease enzyme
(EC 3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the
xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600
XU/g.
[0065] A gluten-reduced dough obtained using the enzymatic composition
comprising: a) a protease enzyme (EC 3.4.23.18); b) a xylanase enzyme (EC
3.2.1.8), and c) water, and wherein the protease enzyme (EC 3.4.23.18) to the
xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1.
[0066] A gluten-reduced dough obtained using the enzymatic composition
comprising: a) a protease enzyme (EC 3.4.23.18); b) a xylanase enzyme (EC
3.2.1.8), and c) at least one excipient, and wherein the protease enzyme (EC
3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to
18:1.
[0067] A gluten-reduced dough obtained using the enzymatic composition
comprising: a) a protease enzyme (EC 3.4.23.18); b) a xylanase enzyme (EC
3.2.1.8), c) water, and d) at least one excipient, wherein the protease enzyme (EC
3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to
18:1.
[0068] A baked product obtained using the enzymatic composition comprising: a)
a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein
the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w
ratio in a range of 4:1 to 18:1.
[0069] A baked product obtained using the enzymatic composition comprising: a)
a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein

the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w
ratio in a range of 4:1 to 18:1, and wherein the protease enzyme (EC 3.4.23.18) has
an enzyme activity in a range of 8500-11500 PCu/g, and the xylanase enzyme (EC
3.2.1.8) has an enzyme activity in a range of 3400-4600 XU/g
[0070] A baked product obtained using the enzymatic composition comprising: a)
a protease enzyme (EC 3.4.23.18); b) a xylanase enzyme (EC 3.2.1.8), and c)
water, and wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme
(EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1.
[0071] A baked product obtained using the enzymatic composition comprising: a)
a protease enzyme (EC 3.4.23.18); b) a xylanase enzyme (EC 3.2.1.8), and c) at
least one excipient, and wherein the protease enzyme (EC 3.4.23.18) to the
xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1.
[0072] A baked product obtained using the enzymatic composition comprising: a)
a protease enzyme (EC 3.4.23.18); b) a xylanase enzyme (EC 3.2.1.8), c) water,
and d) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the
xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1.
[0073] In an embodiment of the present disclosure, there is provided a process for
preparing a composition comprising the steps of: a) obtaining the protease enzyme
(EC 3.4.23.18); b) obtaining the xylanase enzyme (EC 3.2.1.8); and c) contacting
the protease enzyme (EC 3.4.23.18) with the xylanase enzyme (EC 3.2.1.8) to
obtain the composition.
[0074] In an embodiment of the present disclosure, there is provided a process for
preparing a composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a
xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the
xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, said
process comprising the steps of a) obtaining the protease enzyme (EC 3.4.23.18);
b) obtaining the xylanase enzyme (EC 3.2.1.8); and c) contacting the protease
enzyme (EC 3.4.23.18) with the xylanase enzyme (EC 3.2.1.8) to obtain the
composition.
[0075] In an embodiment of the present disclosure, there is provided a process for
preparing a composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a

xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600 XU/g, said process comprising the steps of a) obtaining the protease enzyme (EC 3.4.23.18); b) obtaining the xylanase enzyme (EC 3.2.1.8); and c) contacting the protease enzyme (EC 3.4.23.18) with the xylanase enzyme (EC 3.2.1.8) to obtain the composition.
[0076] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition, said process comprising the steps of a) obtaining the protease enzyme (EC 3.4.23.18); b) obtaining the xylanase enzyme (EC 3.2.1.8); and c) contacting the protease enzyme (EC 3.4.23.18) with the xylanase enzyme (EC 3.2.1.8) to obtain the composition.
[0077] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising: a) a protease enzyme (EC 3.4.23.18); and b) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600 XU/g, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition, said process comprising the steps of a) obtaining the protease enzyme (EC 3.4.23.18); b) obtaining the xylanase enzyme (EC 3.2.1.8); and c) contacting the protease enzyme (EC 3.4.23.18) with the xylanase enzyme (EC 3.2.1.8) to obtain the composition.

[0078] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); and ii) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry; and c) incubating the slurry at a temperature in a range of 3 8°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough. In another embodiment of the present disclosure, incubating the slurry at a temperature in a range of 40°C to 45°C for a time period in a range of 22-26 hours, to obtain a gluten-reduced dough. [0079] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), and iii) water, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough.
[0080] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); and ii) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition; b) contacting the composition with a dough to obtain a slurry; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough.
[0081] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a)

obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), and iii) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough. [0082] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), and iii) water, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition; b) contacting the composition with a dough to obtain a slurry; and c) incubating the slurry at a temperature in a range of 38°Cto 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough.
[0083] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), iii) water, and iv) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough. [0084] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), iii) water, and iv) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; and wherein the protease enzyme (EC 3.4.23.18)

has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition; b) contacting the composition with a dough to obtain a slurry; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough.
[0085] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); and ii) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough. In another embodiment of the present disclosure, the flour and water having a w/v ratio of the flour to water in a range of 1:3.5 to 1:4.5.
[0086] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), and iii) water, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough. In another embodiment of the present disclosure, the flour and water having a w/v ratio of the flour to water in a range of 1:3.5 to 1:4.5.
[0087] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); and ii) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the

xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough.
[0088] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), and iii) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5; and c) incubating the slurry at a temperature in a range of 38°Cto 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough.
[0089] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), and iii) water, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough.

[0090] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), iii) water, and iv) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough.
[0091] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), iii) water, and iv) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough. [0092] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), and iii) water, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5; and wherein the flour is selected from a group consisting of wheat flour, barley flour, rye flour, spelt flour, triticale, and combinations thereof,

and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough. [0093] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); and ii) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5, and wherein the flour is selected from a group consisting of wheat flour, barley flour, rye flour, spelt flour, triticale, and combinations thereof; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough.
[0094] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), and iii) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5, and wherein the flour is selected from a group consisting of wheat flour, barley flour, rye flour, spelt flour, triticale, and combinations thereof; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough. In another embodiment of the present disclosure, the flour is wheat flour. [0095] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a

xylanase enzyme (EC 3.2.1.8), and iii) water, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5, and wherein the flour is selected from a group consisting of wheat flour, barley flour, rye flour, spelt flour, triticale, and combinations thereof; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough.
[0096] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), iii) water, and iv) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5, and wherein the flour is selected from a group consisting of wheat flour, barley flour, rye flour, spelt flour, triticale, and combinations thereof; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough.
[0097] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), iii) water, and iv) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition; b)

contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5, and wherein the flour is selected from a group consisting of wheat flour, barley flour, rye flour, spelt flour, triticale, and combinations thereof; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough.
[0098] In an embodiment of the present disclosure, there is provided a process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), iii) water, and iv) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4.33:1 - 15:1; and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water is 1:4, and wherein the flour is wheat flour; and c) incubating the slurry at a temperature in a range of 40°Cto 45°Cfor a time period in a range of 22-24 hours, to obtain a gluten-reduced dough.
[0099] In an embodiment of the present disclosure, there is provided a process for
reducing gluten content in a dough, said process comprising the steps of: a)
obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a
xylanase enzyme (EC 3.2.1.8), and iii) water, wherein the protease enzyme (EC
3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1
- 18:1; b) contacting the composition with a dough to obtain a slurry; and c)
incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in
a range of 20-28 hours, to obtain a gluten-reduced dough; and wherein the gluten
content is reduced in a range of 18-25% with respect to the dough.
[00100] In an embodiment of the present disclosure, there is provided a
process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii)

a xylanase enzyme (EC 3.2.1.8), iii) water, and iv) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough; and wherein the gluten content is reduced in a range of 18-25% with respect to the dough.
[00101] In an embodiment of the present disclosure, there is provided a
process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); and ii) a xylanase enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition, and wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough; and wherein the gluten content is reduced in a range of 18-25% with respect to the dough.
[00102] In an embodiment of the present disclosure, there is provided a
process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), and iii) at least one excipient being flour selected from a group consisting of wheat flour, barley flour, rye flour, spelt flour, triticale, and combinations thereof, and wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough; and wherein the gluten content is reduced in a range of 18-25% with respect to the dough. In another embodiment of

the present disclosure, the gluten content is reduced in a range of 19-24% with respect to the gluten content in the dough.
[00103] In an embodiment of the present disclosure, there is provided a
process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), iii) water, and iv) at least one excipient being flour selected from a group consisting of wheat flour, barley flour, rye flour, spelt flour, triticale, and combinations thereof, and wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1
- 18:1; b) contacting the composition with a dough to obtain a slurry; and c)
incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in
a range of 20-28 hours, to obtain a gluten-reduced dough; and wherein the gluten
content is reduced in a range of 18-25% with respect to the dough.
[00104] In an embodiment of the present disclosure, there is provided a
process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), iii) water, and iv) at least one excipient being flour selected from a group consisting of wheat flour, barley flour, rye flour, spelt flour, triticale, and combinations thereof, and wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1
- 18:1; b) contacting the composition with a dough to obtain a slurry, wherein the
dough comprises a flour and water having a w/v ratio of the flour to water in a
range of 1:3 to 1:5; and c) incubating the slurry at a temperature in a range of 38°C
to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced
dough; and wherein the gluten content is reduced in a range of 18-25%) with respect
to the dough.
[00105] In an embodiment of the present disclosure, there is provided a
process for reducing gluten content in a dough, said process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), iii) water, and iv) at least one excipient being flour selected from a group consisting of wheat flour, barley flour, rye flour, spelt flour,

triticale, and combinations thereof, and wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain a gluten-reduced dough; and wherein the gluten content is reduced in a range of 18-25% with respect to the dough.
[00106] In an embodiment of the present disclosure, there is provided a
gluten-reduced dough obtained by a process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8) and iii) water, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, b) contacting the composition with a dough to obtain a slurry; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain the gluten-reduced dough.
[00107] In an embodiment of the present disclosure, there is provided a
gluten-reduced dough obtained by a process comprising the steps of: a) obtaining a
composition comprising i) a protease enzyme (EC 3.4.23.18); and ii) a xylanase
enzyme (EC 3.2.1.8), wherein the protease enzyme (EC 3.4.23.18) to the xylanase
enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1, and wherein the
protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and
the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with
respect to the composition; b) contacting the composition with a dough to obtain a
slurry; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a
time period in a range of 20-28 hours, to obtain the gluten-reduced dough.
[00108] In an embodiment of the present disclosure, there is provided a
gluten-reduced dough obtained by a process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase

enzyme (EC 3.2.1.8), and iii) at least one excipient, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry; and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours, to obtain the gluten-reduced dough.
[00109] In an embodiment of the present disclosure, there is provided a
gluten-reduced dough obtained by a process comprising the steps of: a) obtaining a
composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase
enzyme (EC 3.2.1.8), iii) water and iv) at least one excipient, wherein the protease
enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the
range of 4:1 - 18:1; b) contacting the composition with a dough to obtain a slurry;
and c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time
period in a range of 20-28 hours, to obtain the gluten-reduced dough.
[00110] In an embodiment of the present disclosure, there is provided a
gluten-reduced dough obtained by a process comprising the steps of: a) obtaining a composition comprising i) a protease enzyme (EC 3.4.23.18); ii) a xylanase enzyme (EC 3.2.1.8), iii) water and iv) at least one excipient being wheat flour, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in the range of 4.33:1 - 15:1; b) contacting the composition with a dough to obtain a slurry, wherein the dough comprises wheat flour and water having a w/w ratio of the wheat flour to water of 1:4; and c) incubating the slurry at a temperature in a range of 38°Cto 47°Cfor a time period in a range of 20-28 hours, to obtain the gluten-reduced dough, wherein the gluten content is reduced in a range of 20.6%-23.43% with respect to the dough.
[00111] In an embodiment of the present disclosure, the enzymatic
composition of the present disclosure is capable of reducing gluten content in a wide variety of cereal grain flours such as, wheat flour, barley flour, rye flour, spelt flour, triticale, and the like; and food products, such as pasta, energy bars, semolina, and bakery products. EXAMPLES

[00112] The disclosure will now be illustrated with working examples,
which is intended to illustrate the working of disclosure and not intended to take
restrictively to imply any limitations on the scope of the present disclosure. Unless
defined otherwise, all technical and scientific terms used herein have the same
meaning as commonly understood to one of ordinary skill in the art to which this
disclosure belongs. Although methods and materials similar or equivalent to those
described herein can be used in the practice of the disclosed methods and
compositions, the exemplary methods, devices and materials are described herein.
It is to be understood that this disclosure is not limited to particular methods, and
experimental conditions described, as such methods and conditions may apply.
[00113] As mentioned previously, conventionally methods to reduce gluten
are based on avoiding or restricting foods that contain gluten. However, given the vast majority of foods that contain gluten, abstaining from such food products is a quite challenging. Also, currently used gluten substitutes are a compromise on texture, taste and/or consistency. Conventionally used methods to make reduced-gluten wheat flour are time consuming, cost ineffective, temperature and pH specific involving multimodal steps. The present invention is directed towards the use of an enzymatic composition to produce reduced gluten wheat dough. Example 1
Wheat sour dough fermentation
[00114] For this purpose, 1:4 ratio of wheat flour (Aashirvaad MP, ITC) was
added to 4 parts of drinking water (Bisleri). A dose of lOOppm of protease and xylanase enzymes obtained from Advanced Enzyme Technology Ltd. were added to the above slurry. The mixture was placed for incubation for 24 hours at 42°C. Example 2
Preparation of samples and reagents towards quantification by S-ELISA
[00115] The sample preparation protocol was followed as per
manufacturer's instructions (R-Biopharm). Briefly, 0.25g or 250ul of homogenized test sample was weighed and 2.5 ml of cocktail solution (R-Biopharm) was added and mixed well. The same was incubated for 40 minutes at 50°C. The sample is let to cool down and mixed with 7.5 ml 80 % ethanol. The vial is closed and shaken

for 1 h up-side down at room temperature, centrifuged for 10 minutes at 2500g at room temperature. Supernatant was collected in a screw capped vial. lOOul of the same immediately used for assay. For preparation of reagents, all reagents brought to room temperature before use. The sample diluent was diluted with 4 times distilled water. The antibody enzyme conjugate was diluted with 10 times distilled water. The washing buffer was diluted with 9 times distilled water. Example 3
Quantification of gluten by S-ELISA
[00116] 100 ul test samples and standard solutions (Oppb, 5ppb, lOppb,
20ppb, 40ppb, and 80ppb gliadin solution) added to separate wells in duplicates. The test samples and the standard solutions were incubated for 30 minutes at room temperature. The liquid poured out of the wells and the wells were further washed with 250 ul diluted washing buffer. The washing step was repeated for two more times. Following the above, 100 ul of the diluted enzyme conjugate added to each well and incubated for 30 minutes at room temperature. On completion, the liquid was poured out of the wells and vigorously (three times in a row) washed as mentioned above. Further, 50 ul of substrate and 50 ul of chromogen was added to each well. The plate was gently shaken manually to mix the reagents, and the plate was further incubated for 30 min at room temperature in dark. On completion of the above, 100 ul of the stop reagent to each well. The plate was again shaken manually, and the absorbance was measured at 450 nm and read within 30 minutes. The gliadin concentration in ug/kg (ppb) is read from calibration curve and further multiplied by dilution factor. This result is then multiplied by 2 to obtain the gluten concentration (gliadin usually represents 50 % of the proteins present in gluten). Example 4
Screening of bioactives towards reduction in gluten in wheat
[00117] Five enzymes PDZYME PT 100, PDZYME FA 400 and protease
and xylanase were screened at lOOppm concentrations each. A combination of protease to xylanase at a ratio of 3:1 was also tested. The first two enzymes obtained from P D Navkar (Bangalore), the next two enzymes obtained from Advanced Enzymes (Mumbai). The enzymes were screened as per the protocol

presented in examples 1-3. The results are presented in Figure 1. The percentage reduction in gluten were compared to that of the control. From the figure 1 it can be observed that, from among the 4 enzymes that were screened for gluten reduction in wheat flour, the combination of protease and xylanase in a ratio of 3:1, and protease, both at a combined concentration of lOOppm were found to exhibit maximum reduction in gluten, of about 12.88% and 12.32% respectively, in comparison to the control. Example 5
Dose curve of finalized enzymes
[00118] Protease and xylanase, were chosen for their higher activity in
reducing gluten in wheat flour; and a dose curve study was performed for the same from 18.75, 37.75, 40, 56.25, 81.25, 87.5, 93.75, 100 and 200 ppm of protease was used to determine the effect of protease on gluten reduction in wheat flour. These enzymes were screened for dose response as per Example 1-3. The results are presented in Figure 2. Quantitative analysis showed that the percentage reduction in gluten was found to be dose dependent when a concentration of 56.25 ppm of protease, as observed in Figure 2. Maximum reduction was found to be for the concentrations lOOppm and 200ppm, respectively. While lOOppm of protease reduced gluten content in wheat flour by 13.65%>, 200 ppm of protease reduced gluten content in wheat flour by 13.71%), in comparison to the control. Since the difference in percentage reduction of gluten was only marginal between the concentrations lOOppm and 200 ppm; lOOppm was selected as the concentration for the enzyme to reduce gluten. Similar findings were obtained with enzyme xylanase. Concentrations ranging from 6.25, 12.5, 18.75, 25, 50, 100 and 200 ppm of xylanase was used to determine the effect of xylanase on gluten reduction in wheat flour. These enzymes were screened for dose response as per Example 1-3. The results are presented in Figure 3. Quantitative analysis of figure 3 reveals that the percentage reduction was dose dependent. Maximum reduction was found to be for the concentrations lOOppm and 200ppm, respectively. While lOOppm of xylanase reduced gluten content in wheat flour by 8.32%, 200 ppm of xylanase reduced gluten content in wheat flour by 9.64%>, in comparison to the control.

Since the difference in percentage reduction of gluten was only marginal between
the concentrations lOOppm and 200 ppm; lOOppm was selected as the
concentration for the enzyme to reduce gluten.
Example 6
Evaluation of combination of protease and xylanase at various w/w ratios on
reduction of gluten in wheat flour
[00119] Based on the results obtained, effect of combinations of protease
and xylanase in various w/w rations was tested, wherein the maximum individual
concentration of the actives was lOOppm for either of enzymes. The ratios and
concentrations tested are mentioned below in Table 1:
[00120] For the various w/w ratios tested, unexpectedly and surprisingly, the
combinatorial effect was more than the effect of either ingredient alone, the combinatorial effect being synergistic for the ratios. From a combined reading of Table 1, Figure 2, Figure 3, and Figure 4, it is observed that when the actives were used in combination at different ratios, the two actives in combination exhibit a synergistic effect, particularly at ratios of 81.25:18.75, 87.5:12.5, and 96.875:3.125. The inhibition was found to be maximum for the ratios of 78.125:21.875 and 96.875:3.125. It was found to be 23.43% and 20.60% respectively. Advantages of the present disclosure
[00121] The present disclosure reveals an enzymatic composition to reduce
gluten in a wide variety of cereal flours such as wheat flour, barley flour, rye flour,

spelt flour, and other food products containing gluten. The reduced gluten wheat flour produced in the present invention may be used as a 1-for-l substitute in any conventional wheat flour recipes, thus eliminating the necessity of using exclusively gluten-free recipes for gluten sensitive individuals. The flour produced thus can be used to make batter, roti's, cookies, cakes, muffins, sauces, and other baked products.

I/We Claim:
1. An enzyme composition comprising:
a) a protease enzyme (EC 3.4.23.18); and
b) a xylanase enzyme (EC 3.2.1.8),
wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4:1 to 18:1.
2. The composition as claimed in claim 1, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio in a range of 4.33:1 to 15:1.
3. The composition as claimed in claim 1, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio of 6.4:1.
4. The composition as claimed in claim 1, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio of 7:1.
5. The composition as claimed in claim 1, wherein the protease enzyme (EC 3.4.23.18) to the xylanase enzyme (EC 3.2.1.8) has a w/w ratio of 15:1.
6. The composition as claimed in claim 1, wherein the protease enzyme (EC 3.4.23.18) has an enzyme activity in a range of 8500-11500 PCu/g, and the xylanase enzyme (EC 3.2.1.8) has an enzyme activity in a range of 3400-4600 XU/g.
7. The composition as claimed in claim 1, wherein the composition further comprises water.
8. The composition as claimed in any one of the claims 1-7, wherein the protease enzyme (EC 3.4.23.18) has a concentration in a range of 80-95 ppm, and the xylanase enzyme (EC 3.2.1.8) has a concentration in a range of 5-20 ppm with respect to the composition.
9. The composition as claimed in any one of the claims 1-8, wherein the composition further comprises at least one excipient.
10. A process for preparing the composition as claimed in claim 1, said method comprising:
a) obtaining the protease enzyme (EC 3.4.23.18);

b) obtaining the xylanase enzyme (EC 3.2.1.8); and
c) contacting the protease enzyme (EC 3.4.23.18) with the xylanase enzyme (EC 3.2.1.8) to obtain the composition.
11. A process for reducing gluten content in a dough, said process comprising:
a) obtaining the composition as claimed in any one of the claims 7-9;
b) contacting the composition with a dough to obtain a slurry; and
c) incubating the slurry at a temperature in a range of 38°C to 47°C for a time period in a range of 20-28 hours,
to obtain a gluten-reduced dough.
12. The process as claimed in claim 11, wherein the dough comprises a flour and water having a w/v ratio of the flour to water in a range of 1:3 to 1:5.
13. The process as claimed in claim 12, wherein the flour is selected from a group consisting of wheat flour, barley flour, rye flour, spelt flour, triticale, and combinations thereof.
14. The process as claimed in claim 11, wherein the gluten content is reduced in a range of 18-25% with respect to the gluten content in the dough.
15. A gluten-reduced dough obtained using the process as claimed in claim 11.
16. A gluten-reduced dough obtained using the composition as claimed in claims 1-9.
17. A baked product obtained using the composition as claimed in claims 1-9.