FIELD OF THE INVENTION
[001] The present disclosure relates to the field of food processing. In particular, the present disclosure relates to a method for reducing the content of gluten in wheat flour.
BACKGROUND OF THE INVENTION
[001] Gluten is a composite protein comprising of glutenin and gliadin, found in
wheat, barley, oat and rye. It constitutes more than 75% of total protein in wheat
bread. Gluten provides the viscoelasticity to kneaded dough and is responsible for the
chewy and elastic texture.
[002] Glutenin and gliadin are also considered as toxic proteins of the wheat and are
responsible for conditions such as celiac disease, wheat allergy and non-celiac gluten
sensitivity (Anna Sapone et.al., BMC Medicine, 2012, 10:13). Celiac disease, an
autoimmune disorder, is one of the most adverse diseases associated with gluten
consumption and affects about 1-2% of the general population (Lundin KE et.al.,
2015, 12(9): 507-15). Individuals suffering from celiac disease are intolerant towards
gluten protein and consumption of the same causes disruption in the lining of the
intestine.
[003] A unique pathophysiological response is observed to gluten ingestion with
each gluten related disorder. These individuals generally experience an inflammatory
autoimmune cascade which may lead to enteropathy and malabsorption syndrome
(Elli et. Al., World J gastroenterol. 2015, 21(23): 7110-7119).
[004] Due to increasing incidences of gluten intolerance, gluten free products have
gained popularity and are conquering the food market with their products.
[005] US20100297323A1 reveals a composition of gluten free flour comprising
microalgal mass which reduces at least one symptom of gluten intolerance in
subjects.
[006] Currently the process employed for the preparation of various gluten free
products comprises the use of array of microorganisms. Although food products with

reduced gluten content are available to alleviate the levels of severe allergy in patients suffering from gluten intolerance, the method for preparation are cost ineffective, time consuming and involves multiple steps and components. Thus, there is need for more efficient processes for gluten reduction that are both economical and effective.
SUMMARY OF THE INVENTION
[007] In an aspect of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second mixture at a
o
temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein the reduction of gluten in the flour is in the range of 70-80%.
[008] In an aspect of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining wheat flour; (b) adding water to the wheat flour at a w/v ratio of 1:4 of wheat flour to water to obtain a first mixture; (c) adding 200ppm of SeBake PP to the first mixture to
o
obtain a second mixture; (d) incubating the second mixture at a temperature of 42 C for a time period of 24 hours; and (e) obtaining a wheat dough with reduced gluten content, wherein the reduction of gluten in the flour is in the range of 70-80%. [009] 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 ACCOMPANYING DRAWINGS

[010] 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 understood by reference to the drawings in combination with the detailed
description of the specific embodiments presented herein.
[Oil] Figure 1 represents the effect of 200ppm concentrations of five enzymes with
respect to percentage reduction of gluten in wheat flour, in accordance with an
embodiment of the present disclosure.
[012] Figure 2 depicts the effect of various concentrations of SeBake PP protease
towards reduction of gluten in wheat flour, in accordance with an embodiment of the
present disclosure.
[013] Figure 3 represents the change in percentage reduction of gluten content at
different time points, in accordance with an embodiment of the present disclosure.
[014] Figure 4 depicts the change in percentage gluten content at different
temperatures, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[015] 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
[016] 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.

[017] 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.
[018] 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".
[019] 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.
[020] The term "including" is used to mean "including but not limited to".
"Including" and "including but not limited to" are used interchangeably.
[021] 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.
[022] Gluten is the structural protein comprising 75-85% of the total protein content
in wheat and associated grains. It is co joined with starch in the endosperm of various
grass related grains. Wheat allergy, celiac disease and non-celiac gluten sensitivity
are health conditions related to the intolerance towards gluten consumption.
[023] Proteases belong to class of enzyme that aid in and accelerate the digestion of
various proteins. They catalyse proteolysis i.e. hydrolysis of peptide bond and
converts protein to smaller peptides or even amino acids.
[024] Gluten intolerance are observed in individuals with genetic predisposition
towards the disease. Upon exposure to the protein an abnormal immune response is
observed which triggers the release of autoantibodies that effects multiple organs
(Lundin KE et. al., Nat Rev Gastroenterol Hepatol. 2015, 12(9): 507-15). Wheat
which is a staple food grain is widely consumed by a large population, contains about

80% of gluten. Patients suffering from gluten intolerance are advised to follow a strict gluten free diet. The processes currently available for preparation of such food products employ use of more than one microorganisms as well as enzymes from fungal origin, resulting in a higher turn over time for processing. Further, the acceptability of these enzymes is often low due to their microbial origin. The present disclosure, provides a process for reduction of gluten content in wheat flour by employing plant based protease. SeBake PP is a protease derived from latex of immature papaya fruit containing 500000 tyrosine units/g. The enzyme has superior ability in reducing the gluten content by proteolysis when compared to other proteases of fungal and bacterial origin. In addition, the acceptability of the SeBake PP consumption in food products is more due to its plant origin. The subject matter of the present disclosure provides an efficient and more acceptable method of gluten reduction in flour. The process therefore provides a solution to the problem of increased gluten intolerance among the population, as it can be used to obtain flour-based products with highly reduced gluten levels.
[025] 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. [026] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein the reduction of gluten in the flour is in the range of 70-80%.
[027] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a

protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein the dough obtained in step (e) is freeze-dried to obtain a low-gluten flour, and the reduction of gluten in the flour is in the range of 70-80%.
[028] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein said flour is selected from the group consisting of wheat flour, barley flour, rye flour and combinations thereof, and the reduction of gluten in the flour is in the range of 70-80%.
[029] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein incubating the second mixture in step (d) is done at a rotation speed in the range of 150-300 RPM, and the reduction of gluten in the flour is in the range of 70-80%). [030] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein incubating

the second mixture in step (d) is done at a rotation speed of 200 RPM, and the reduction of gluten in the flour is in the range of 70-80%.
[031] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein said water used includes, but is not limited to, tap water, Bisleri water, distilled water, double distilled water, mineral water, and the reduction of gluten in the flour is in the range of 70-80%.
[032] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein said water is Bisleri water, and the reduction of gluten in the flour is in the range of 70-80%). [033] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein said flour is wheat flour, and the reduction of gluten in the flour is in the range of 70-80%). [034] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second

mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein said flour is barley flour, and the reduction of gluten in the flour is in the range of 70-80%. [035] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein said flour is rye flour, and the reduction of gluten in the flour is in the range of 70-80%. [036] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein said first mixture comprises the flour and water at a w/v ratio in the range of 1:3-1:5, and the reduction of gluten in the flour is in the range of 70-80%).
[037] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein said first mixture comprises the flour and water at a w/v ratio of 1:4, and the reduction of gluten in the flour is in the range of 70-80%.
[038] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining wheat flour; (b) adding water to the wheat flour at a w/v ratio in the range

of 1:3-l :5 of wheat flour to water to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second mixture at a
o
temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein the reduction of gluten in the flour is in the range of 70-80%.
[039] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining wheat flour; (b) adding water to the wheat flour at a w/v ratio of 1:4 of wheat flour to water to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second mixture at a temperature in the
o
range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein the reduction of gluten in the flour is in the range of 70-80%.
[040] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein the protease is SeBake PP, and the reduction of gluten in the flour is in the range of 70-80%). [041] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein the protease is added at a concentration in the range of 200 ppm-300 ppm, and the reduction of gluten in the flour is in the range of 70-80%.

[042] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding 200-300ppm of SeBake PP to the first mixture to obtain a second mixture; (d) incubating
o
the second mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein said flour is selected from the group consisting of wheat flour, barley flour, rye flour and combinations thereof, and the reduction of gluten in the flour is in the range of 70-80%.
[043] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding 200-300ppm of SeBake PP to the first mixture to obtain a second mixture; (d) incubating
o
the second mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein incubating the second mixture in step (d) is done at a rotation speed of 150-300 RPM, and the reduction of gluten in the flour is in the range of 70-80%. [044] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding 200-300ppm of SeBake PP to the first mixture to obtain a second mixture; (d) incubating
o
the second mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein incubating the second mixture in step (d) is done at a rotation speed of 200 RPM, and the reduction of gluten in the flour is in the range of 70-80%).
[045] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour at a w/v ratio in the range of 1:3-l :5 of flour to water to obtain a first mixture; (c) adding 200-3OOppm of SeBake PP to the

first mixture to obtain a second mixture; (d) incubating the second mixture at a
o
temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein the reduction of gluten in the flour is in the range of 70-80%.
[046] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour at a w/v ratio in the range of 1:3-l :5 of flour to water to obtain a first mixture; (c) adding 200-3OOppm of SeBake PP to the first mixture to obtain a second mixture; (d) incubating the second mixture at a
o
temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein incubating the second mixture in step (d) is done at a rotation speed of 150- 300 RPM, and the reduction of gluten in the flour is in the range of 70-80%.
[047] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining wheat flour; (b) adding water to the wheat flour at a w/v ratio of 1:4 of wheat flour to water to obtain a first mixture; (c) adding 200-300ppm of SeBake PP to the first mixture to obtain a second mixture; (d) incubating the second mixture at a
o
temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein the reduction of gluten in the flour is in the range of 70-80%.
[048] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein the protease is added at a concentration of 200 ppm, and the reduction of gluten in the flour is in the range of 70-80%.

[049] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding SeBake PP to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein SeBake PP is added at a concentration of 200 ppm, and the reduction of gluten in the flour is in the range of 70-80%.
[050] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining wheat flour; (b) adding water to the wheat flour at a w/v ratio of 1:4 of wheat flour to water to obtain a first mixture; (c) adding 200ppm of SeBake PP to the first mixture to obtain a second mixture; (d) incubating the second mixture at a
o
temperature in the range of 42-45 C for a time period in the range of 24-48 hours; and (e) obtaining a dough with reduced gluten content, wherein the reduction of gluten in the flour is in the range of 70-80%.
[051] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature in the range of 42-45 C for a time period of 24 hours; and (e) obtaining a dough with reduced gluten content, wherein the reduction of gluten in the flour is in the range of 70-80%.
[052] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature of 42 C for a time period in the range of 24-48 hours; and

(e) obtaining a dough with reduced gluten content, wherein the reduction of gluten in the flour is in the range of 70-80%.
[053] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining flour; (b) adding water to the flour to obtain a first mixture; (c) adding a protease to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature of 42 C for a time period of 24 hours; and (e) obtaining a dough with reduced gluten content, wherein the reduction of gluten in the flour is in the range of 70-80%.
[054] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining wheat flour; (b) adding water to the wheat flour at a w/v ratio in the range of 1:3-1:5 of wheat flour to water to obtain a first mixture; (c) adding 200-300ppm of SeBake PP to the first mixture to obtain a second mixture; (d) incubating the second
o
mixture at a temperature of 42 C for a time period of 24 hours; and (e) obtaining a dough with reduced gluten content, wherein the reduction of gluten in the flour is in the range of 70-80%.
[055] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining wheat flour; (b) adding water to the wheat flour at a w/v ratio of 1:4 of wheat flour to water to obtain a first mixture; (c) adding 200-300ppm of SeBake PP to the first mixture to obtain a second mixture; (d) incubating the second mixture at a
o
temperature of 42 C for a time period of 24 hours; and (e) obtaining a dough with reduced gluten content, wherein incubating the second mixture in step (d) is done at a rotation speed of 150-300 RPM, and the reduction of gluten in the flour is in the range of 70-80%.
[056] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining wheat flour; (b) adding water to the wheat flour at a w/v ratio of 1:4 of

wheat flour to water to obtain a first mixture; (c) adding 200 ppm of SeBake PP to the first mixture to obtain a second mixture; (d) incubating the second mixture at a
o
temperature of 42 C for a time period of 24 hours; and (e) obtaining a dough with reduced gluten content, wherein incubating the second mixture in step (d) is done at a rotation speed of 200 RPM, and the reduction of gluten in the flour is in the range of 70-80%.
[057] In an embodiment of the present disclosure, there is provided a process for reduction of gluten content in flour, said process comprising the steps of: (a) obtaining wheat flour; (b) adding water to the wheat flour at a w/v ratio of 1:4 of wheat flour to water to obtain a first mixture; (c) adding 200ppm of SeBake PP to the first mixture to obtain a second mixture; (d) incubating the second mixture at a
o
temperature of 42 C for a time period of 24 hours; and (e) obtaining a wheat dough with reduced gluten content, wherein the reduction of gluten in the flour is in the range of 70-80%.
[058] In an embodiment of the present disclosure, there is provided a dough with reduced gluten content obtained from a process described herein, wherein said dough can be freeze dried to obtain a flour with reduced gluten content. [059] In an embodiment of the present disclosure, there is provided a flour with reduced gluten content obtained from a process described herein, wherein said flour can be used in preparation of low- gluten content food products. [060] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.
EXAMPLES

[061] 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 vary.
Example 1
Materials and Methods
[062] Wheat flour used in the study is Aashirwaad MP Atta (ITC). The enzyme
SeBake PP, SeBakeNP and SeBake crisp plus were procured from Advanced Enzyme
technology limited (Mumbai) and enzymes PDZYME PT 100 and PDZYME FA 250
were procured from P D Navkar, Biochem pvt limited (Bangalore).
[063] Preparation of the wheat dough: 80g of wheat flour (having moisture content
of approximately 9%) was mixed with 320g of water (Bisleri) i.e. in a w/w ratio of
1:4. This ratio was found to be suitable for preparation of dough with acceptable
consistency and texture.
[064] Screening of proteases for reduction of gluten in wheat dough: Five enzymes
viz., PDZYME PT 100, PDZYME FA, SeBake PP, SeBakeNP and SeBake crisp plus
were added separately to the wheat dough prepared earlier at a concentration of
200ppm each (Figure 1). Wheat dough without any treatment served as control. The
mixture of enzyme and dough was then placed in a shaking incubator at 42°C for 24
hours at a rotation speed of 200 RPM for fermentation.
[065] Following incubation for the said time-period, the dough was then freeze-
dried to obtain a flour with low gluten content.

[066] Titration of SeBake PP enzyme at different concentrations: In order to identify the absolute concentration of enzyme for gluten reduction, a dose curve study was performed with SeBake PP by adding different concentrations of the enzyme in wheat dough (prepared same as described earlier). The concentrations tested were: 50ppm, lOOppm, 200ppm and 300ppm (Figure 2). Wheat dough without any treatment served as control. The dough was incubated in a shaking incubator at 42°C for 24 hours at a rotation speed of 200 RPM.
[067] Titration of SeBake PP enzyme at different time points: Two mixtures of 200ppm of SeBake enzyme and wheat dough (prepared same as described earlier) were prepared and placed in a shaking incubator at 42°C for 24 hours and 48 hours at a rotation speed of 200 RPM to determine the appropriate time for gluten reduction (Figure 3).
[068] Titration of SeBake PP enzyme at different temperatures:_Mixtures of 200 ppm SeBake PP enzyme and wheat dough (prepared same as described earlier) were prepared and incubated separately in shaking incubator at 37°C, 40°C, 42°C and 45°C for a duration of 24 hours at a rotation speed of 200 RPM (Figure 4).
Example 2
Analysis of reduction in gluten content by S-ELISA:
[069] The kit for measuring the gluten content was procured from R-biopharma. All
the steps for sample preparation and for ELISA were performed as per the
manufacture's protocol.
[070] Preparation of samples: All the samples at various conditions for gluten
reduction tested in example 1 were homogenized. 0.25g of sample by weight or 250ul
of sample by volume of the homogenized samples were added to 2.5ml of cocktail
solution (R-biopharm) and mixed well. The samples were then incubated at 50°C for
40 minutes. Post heating the samples were cooled down and mixed with 80% ethanol.
The samples were then shaken for 1-hour upside down position at room temperature

in a closed vial and centrifuged at 2500g for 10 minutes. The supernatant obtained were collected in screw capped vials and further used for S-ELISA. [071] Protocol of S-ELISA: All the reagents were brought down to room temperature before use. The working solution of the reagents were prepared from the stock solutions in distilled water. Sample diluent was diluted to 1:5, the antibody enzyme conjugate was diluted to 1:11 and the washing buffer was diluted to 1:10. [072] 100 ul of the test samples prepared earlier and 100 ul of standard solutions (Oppb, 5ppb, lOppb, 20ppb, 40ppb and 80ppb gliadin solution) were added to separate wells in duplicates and incubated for 30 minutes at room temperature. After incubation the samples and standards were removed and the wells were washed thrice with 250ul of diluted washing buffer. lOOul of the antibody was added to the washed wells and incubated for 30 minutes at room temperature. The antibody was removed and the wells were washed again with washing buffer. 50ul of substrate along with 50ul of chromogen was added to all the wells, contents were gently mixed by shaking manually and placed at room temperature for 30 minutes in dark. Finally, lOOul of stop solution was added to all the wells and was measured at 450 nm within 30 minutes. The concentration of gliadin in the samples was measured in ug/kg (ppb) with respect to the standard curve and multiplied by the dilution factor. The resulting values were then multiplied by 2 in order to get the final gluten content in the samples (gliadin usually represents 50% of the proteins present in gluten).
Example 3 Results
[073] Figure 1 depicts the comparison amongst various proteases tested for gluten reduction and SeBake PP showed highest reduction, greater than 75% at 200ppm when compared to control dough. PDZYME PT 100 and PDZYME FA 250 showed a reduction of 25% and 40% respectively. Whereas, SeBake NP and SeBake crisp plus reduced gluten by 55% and 10% respectively.

[074] Based on the above data different concentrations of SeBake PP enzyme were tested for gluten reduction. As is evident in Figure 2, a linear reduction was observed from 50ppm to 200ppm. A reduction of 20%, 50%, and 75% was observed with concentrations 50ppm, lOOppm and 200ppm respectively. Post 200ppm an equilibrium stage was reached, wherein there was no further increase in the percentage gluten reduction. Therefore, a concentration of 200ppm was found to be optimum for the process.
[075] The activity of SeBake PP enzyme was tested at two different time points of 24 hours and 48 hours. Similar inhibition in the gluten content i.e. almost 75% was observed (Figure 3) at both the time points.
[076] After confirming the optimum concentration and time of the enzyme a titration over a range of temperature was performed using SeBake PP enzyme. As depicted in Figure 4 The reduction in gluten content was found to be enhanced, almost 75%) at higher temperatures of 42°C and 45°C However, as the temperature was decreased to 40°C and further to 37°C a decline of almost 10% in inhibition of gluten content was observed.
[077] Thus in the present study higher inhibition of gluten content in wheat flour was achieved. The process employs the use of SeBake PP enzyme, which when used at 200ppm, at a temperature of 42°C for a time period of 24 hours was able to reduce the total gluten content by 75% in wheat dough. A marginal increase in the total moisture content was also observed in the flour with reduced gluten. The final moisture content was approximately 10%. The optimum conditions for the process was inferred from the above set of data obtained by performing various studies. Therefore, said process cannot be arrived at without undue experimentation.
Advantages of the present disclosure: Overall, present disclosure provides a process which can be employed to reduce the total content of gluten in flour such as wheat flour, rye flour and barley flour. Additionally, a reduction of greater than 80% can be achieved by use of only one plant based protease enzyme, compared with

other commonly used enzymes, using the method so developed. The disclosed process has superiority over the earlier process in terms of both time and number of participating components. With the proposed process the total time of preparation can be reduced to more than half. The process can be further employed for preparation of reduced gluten and gluten free products for consumption by individuals who suffer from gluten intolerance. Further, the identification of this process for reducing the gluten content in wheat based flour employing less time and fewer components cannot be arrived at without undue experimentation, and is not obvious to a person skilled in the art.

I/We Claim:
1. A process for reduction of gluten content in flour, said process comprising
the steps of:
(a) obtaining flour;
(b) adding water to the flour to obtain a first mixture;
(c) adding a protease to the first mixture to obtain a second mixture;
(d) incubating the second mixture at a temperature in the range of 42- 45°C for a time period in the range of 24-48 hours; and
(e) obtaining a dough with reduced gluten content,
wherein the reduction of gluten in the flour is in the range of 70-80%.
2. The process as claimed in claim 1, wherein said flour is selected from the group consisting of wheat flour, barley flour, rye flour, and combinations thereof.
3. The process as claimed in claim 2, wherein said flour is wheat flour.
4. The process as claimed in claim 1, wherein said first mixture comprises the flour and water at a w/v ratio in the range of 1:3-l :5.
5. The process as claimed in claim 4, wherein said first mixture comprises the
flour and water at a w/v ratio of 1:4.
6. The process as claimed in claim 1, wherein the protease is SeBake PP.
7. The process as claimed in claim 1, wherein the protease is added at a concentration in the range of 200 ppm-300 ppm.
8. The process as claimed in claim 6, wherein the protease is added at a concentration of 200ppm.
9. The process as claimed in claim 1, wherein the second mixture is incubated at a temperature of 42°C for a time period of 24 hours.
10. A process for reduction of gluten content in wheat flour, said process comprising the steps of:

(a) obtaining wheat flour;
(b) adding water to the wheat flour at a w/v ratio of 1:4 of wheat flour to water to obtain a first mixture;
(c) adding 200ppm of SeBake PP to the first mixture to obtain a second mixture;
(d) incubating the second mixture at a temperature of 42°C for a time period of 24 hours;
(e) obtaining a dough with reduced gluten content,
wherein the reduction of gluten in the wheat flour is in the range of 70-80%.