Description:
FIELD OF THE INVENTION:

The present invention relates to a composition that effectively reduces gluten content from gluten containing food substrates. In particular, the present invention relates to a composition comprising surfactant, which reduces the processing time and amount of water requirement for degradation of gluten from gluten containing food substrates in less than three hours.

BACKGROUND OF THE INVENTION:

Gluten is a structural protein found in a few grains such as wheat, barley, rye and triticale (a cross between wheat and rye). Wheat gluten comprises of glutenin (a glutelin) and gliadin (a prolamin). Wheat flour when mixes with water, the glutenin and gliadin form a sticky network having glue-like consistency (named ‘gluten’) and makes the dough elastic, which in turn, gives wheat based product, the ability to provides a chewy, satisfying texture and rise during baking process.

Gliadin is responsible for most of the adverse health effects of gluten. Gliadin is immunogenic and can cause problems like celiac disease (CD), non-celiac gluten sensitivity (NCGS), wheat allergy, and other gluten intolerance symptoms. Celiac disease, estimated to be affecting around 1% population, is an autoimmune disorder in which the body treats gluten as foreign substance. However, it is also being realized that a large population ranging from 0.5 to 13% suffer from a non-celiac gluten sensitivity (NCGS). Non-celiac gluten sensitivity causes some signs and symptoms associated with celiac disease — including abdominal pain, bloating, diarrhoea, constipation, "foggy brain," rash or headache — even though there is no damage to the tissues of the small intestine. Studies show that the immune system plays a role, but the process is not well understood. Gluten ataxia, an autoimmune disorder, affects certain nerve tissues and causes problems with muscle control and voluntary muscle movement.

Currently, there is no approved medication for the treatment of celiac disease. Accordingly, a gluten-free diet is essential for managing signs and symptoms of celiac disease and other medical conditions associated with gluten.

Gluten is widely found in wheat based food products, and therefore removing wheat completely from diet will change overall intake of fiber, vitamins and other nutrients. Maida is a refined white flour made from wheat that is the most widely used in a wide range of edible items that include rotis, nans, confectionary products, cakes, cookies/biscuit, and many packaged food products etc. Maida therefore also happens to be the prime source of glutens found in these items.

There are various methods known in the art for the removal or reduction of gluten from gluten containing food substrates. One such method is employment of enzymes, which naturally break gluten and reduce its amounts to below ppm levels in the final product. The disadvantage in use of enzymes alone is that the process essentially requires a long duration typically 24-48 hours and large quantities of a medium (such as water) to be effective for gluten removal/reduction. This excess water (additional medium) removal makes the entire process uneconomical for commercial utilization and production. Furthermore, microbial cultures including fungal cultures have also been used to reduce levels of gluten in gluten containing grains.

Another solution known in the art is the use of modified enzymes/ engineered enzymes. However, this adds to the total production costs and may not be the best possible solution for all substrates.

On the other hand, the enzymes, and particularly proteases, are commonly used as active ingredients in detergents to improve their efficiency (dos Santos Aguilar & Sato, 2018; Hellmuth & Dreja, 2016), especially in the detergent industry for laundry and dishwasher (Saperas & Fonfría-Subirós, 2011; Vojcic et al., 2015). However, until now, and to our knowledge, no studies have described the inclusion of proteases in detergents for the elimination of gluten in equipment and work surfaces of the food industry.

WO 2016/210408 discloses a method of processing an initial starch containing gluten protein to produce a purified starch having less than 20 parts per million of a gluten protein (i.e., "gluten free"). A slurry of the unpurified starch is treated with an agent to degrade the gluten protein, and then the degraded gluten protein is removed, resulting in a slurry of the purified starch. The agent is selected from among acids, bases, alcohols, surfactants, proteases, chaotropic agents, reducing agents, and combinations thereof. However, in this document, the raw starch with ~470ppm gluten is being processed using a large amount of water to achieve dried starch with < 20ppm of gluten. clearly, there is no reference of using refined wheat flour with gluten content of ~ 7500ppm and how to convert it to a slurry to achieve <20ppm gluten (dry basis) with very less amount of water.

Such methods of prior art require the substrate and enzyme to be in highly dispersed form for degrading the gluten in such substrates. In order to high dispersion, 3 to 4 fold water (v/v ) may be required for carrying out the gluten degradation to <20ppm without the use of surfactant. However, in case of biscuits and several other bakery products, use of large amount of water is impractical particularly for product manufacturing for biscuits, cookies, and cakes among others.

Therefore, there remains a need for a commercially viable and technically usable product and process to prepare the same.

OBJECT OF THE INVENTION

Accordingly, the object of the present invention is to provide a composition for reducing the content of gluten in gluten containing food substrate.

Another object of the present invention is to provide a composition for reducing the content of gluten in gluten containing food substrate, which composition is effective yet simple and cost effective.

Yet another object of the present invention is to provide a method for reducing or removing gluten from gluten containing food substrate.

A further object of the present invention is to provide a time efficient method, which effectively reduces gluten content below 20 ppm from the gluten containing food substrate, which removes gluten in less than 3 hours.

SUMMARY OF THE INVENTION
According to first aspect of present invention, there is provided a pre-mix composition for reducing gluten content in gluten containing food substrates; comprising
at least one enzyme,
at least one surfactant; and
a carrier.

According to second aspect of present invention, there is provided a method of treating a gluten containing food substrate with the premix composition to degrade/reduce the gluten content of the food substrate; said method comprising:
- contacting the premix composition with said gluten containing food substrate to obtain a slurry mixture;
- stirring the slurry mixture at a temperature ranging from 27 oC to about 45 oC at 200 rpm to 900 rpm for a period ranging from 1 to about 24 hours to obtain a slurry with gluten content below 20 ppm.

According to third aspect of present invention, there is provided a food substrate treated by the premix composition, having gluten content below 60 ppm, preferably below 40 ppm and most preferably below 20 ppm.

DESCRIPTION OF THE INVENTION:

The terminology used herein is for the purpose of describing particular various embodiments only and is not intended to be limiting of various embodiments. As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising" used herein specify the presence of stated features, steps, components, and/or groups thereof, but do not preclude the presence or addition of one or more other features, steps, components, and/or groups thereof. Also, expressions such as "at least one of," when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

Described herein is a pre-mix composition for reducing gluten content in gluten containing food substrates. Said pre-mix composition comprises at least one enzyme; at least one surfactant; and a carrier. The present inventors surprisingly found that addition of a small quantity of surfactant to enzyme composition increases the efficiency of the gluten degrading enzyme.

The pre-mix composition comprises
• at least one enzyme;
• at least one surfactant; and
• a carrier

The carrier is present in an amount in the range from 40 % to 70 wt % with respect to the total weight of the pre-mix. Said carrier is selected from the group comprising of water, oil, organic solvents (alcohol, hexane, petroleum ether etc) or combination thereof. Preferably, the carrier comprises water.

The surfactant is selected from the group consisting of Polysorbate-20 (Tween-20), Polysorbate-60 (Tween-60), Polysorbate-80 (Tween-80), sodium stearoyl lactylate, lecithin or combinations thereof.

The enzyme selected from a group consisting of papain, neutral protease, aspartic protease, cysteine protease, broad spectrum endo-protease thereof. The enzymes may be obtained from microbial sources or synthetically prepared. The enzymes can be commercially available or academic interest with commercial development potential.

In an embodiment the ratio of surfactant: enzyme in the premix composition is in the range from 1:2 to 1:10.

In a preferred embodiment, the ratio of surfactant: enzyme in the premix composition is in the range from 1:2 to 1:4.

The substrate includes but is not limited to flours from wheat (such as maida, atta etc.), barley, rye.

The premix composition is capable of degrade/reduce the gluten and its components (gliadin and glutenin) from a predetermined amount of at least one substrate below 60 ppm, preferably below 40 ppm and most preferably below 20 ppm. The composition is capable of degrading/reducing gluten content in less than 3 hours preferably in less than 2.5 hours.

In another embodiment the present invention provides a method of treating the substrate with premix of the present invention to degrade/reduce the gluten content of the gluten containing food substrate; said method comprising:
- contacting the premix composition with a gluten containing food substrate to obtain a slurry mixture;
- stirring the slurry mixture at a temperature ranging from 27 oC to about 45 oC at 200 rpm to 900 rpm for a period ranging from 1 to about 24 hours to obtain a slurry with gluten content below 20 ppm.

In the present invention, the surfactant facilitates the contact of enzyme with the gluten components i.e., gliadin and glutenin in the substrate. Consequently, the process of degradation of the gliadin and glutenin is enhanced. Such fast contact of enzyme (protease) with its substrate (gliadin & glutenin) is able to carry out the process of degradation of gluten in less than three hours, as against the several hours mentions in some prior art. Further, it has further been found that the amount of water required for processing the slurry is about 20% to 60 % lower than the amount required for processing the slurry mixture if it is only treated with enzyme.

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.

EXAMPLES
EXAMPLE-1
Preparation of slurry with reduced level of gluten: Stirring wheat flour and water at a temperature ranging from 27°C to about 45°C at 200 rpm to 900 rpm for a period of about 2 hours) to obtain a slurry with gluten content below 20 ppm as per the below table.

Table-1
Working Premix Examples
Enzyme Tween-20 Tween-80 Water Substrate (Maida) Extra Water added Gluten value of the FG
Unit g g g g G g ppm
1 2 0 0.5 4 100 78 7.26
2 2 0 0.5 4 100 63 6.69
3 1.5 0 0.5 3 100 64 8.72
4 1 0 0.5 2 100 65 11.69

Composition no. 1 is a working example because surfactant to enzyme ratio is between 1:4 carriers in the premix is 61.5%, extra carrier required is 78 ml and the gluten value is 7.26 ppm.

Composition no. 2 is a working example because surfactant to enzyme ratio is between 1:4 carriers in the premix is 61.5%, extra carrier required is 63 ml and the gluten value is 6.69 ppm.

Composition no. 3 is a working example because surfactant to enzyme ratio is between 1:3 carriers in the premix is 60%, extra carrier required is 64 ml and the gluten value is 8.72 ppm.

Composition no. 4 is a working example because surfactant to enzyme ratio is between 1:2 carriers in the premix is 57.1%, extra carrier required is 65 ml and the gluten value is 11.69 ppm.

These working examples are with gluten value less than 20 ppm, with the lesser amount of total carrier and surfactant of about 0.5 gm

Comparative compositions:
Premix compositions were prepared according to the above example without one or more surfactants.

Table-2
Non-Working Premix examples
S/N Enzyme Tween 20 Tween 80 Water Substrate (Maida) Extra Water Added Gluten Content of FG
Units g g g g g g ppm
C1 2 0 0 4 100 96 6.15
C2 2 0.2 0 4 100 96 9.12
C3 1.5 0.2 0 3 100 97 9.4
C4 2 0 0.2 4 100 96 12.58
C5 1.5 0 0.2 3 100 97 12.54
C6 1 0 0 2 100 65 51.7
C7 1 0.2 0 2 100 61 38.9
C8 1.5 0 0 3 90 64 21.1
C9 1.5 0 0.4 3 90 58 21.3
C10 1 0 0.4 2 90 57 25.6
C11 2 0 0.4 4 90 59 20.2
C12 0.5 0 0.5 1 90 62 26.4
C13 6 0 0.5 12 90 50 -
C14 0 0 0 4 100 96 >80

Even though, gluten value in the finished goods are less than the 20 ppm for Composition no. C1 to C5, these examples falls under non-working examples because the required total water content are high, with higher water content prototyping (converting them to a food product such as biscuits or cookies etc) is difficult and sensorially inferior.

Composition no. C6 to C11 falls under non-working examples because the gluten value in the finished goods are more than the 20 ppm.

Composition no. C12 is non-working example because surfactant to enzyme ratio is 1:1 and the gluten value is more than 20 ppm.

Composition no. C13 is non-working example because surfactant to enzyme ratio is 1:12 and the sensory of the product is not palatable and hence gluten value not analyzed.

EXAMPLE-2
Preparation of biscuits using the slurry mixture treated with premix composition of Example 1 (Premix Composition is provided in Table-1):

As per the examples, the biscuit prepared from the premix given in the “working premix table” are able to degrade/reduce gluten content less than 20 ppm in 2.0 hours. Specifically, the biscuit prepared from the slurry with 100 gm of Maida, 67ml water content, in 2 gm of enzymes and tween 80 (0.5 gm) shows synergistic effect in bringing down the gluten value when compared to that of biscuit prepared from the slurry with 100 gm of Maida, 67ml water content in 2 gm of enzymes without tween 80.

EXAMPLE-3
Slurry Preparation: Gluten Free Maida processing with surfactant.
• Briefly, the required water volume (100ml, 82ml & 67ml) in a glass beaker placed in a water bath, while maintaining the temperature at around 40 °C.
• The enzyme was added with different concentration (2%, 1.5% and 1%) respectively in portions to the pre-heated water under constant stirring using an overhead stirrer (800-900 rpm).
• Surfactants, Tween 20 (@ 0.2% & 0.5%) and Tween 80 (@ 0.2% & 0.5%) were added into the enzyme water and allowed stirring for 3-5mins.
• Then the Maida flour was introduced in portions into the mixer solution. The mixture was allowed to stir for 2 hours at 800-900 rpm.
• Once the process was complete, the slurry was used for making the biscuits.

• Slurry Composition table:
Table-3:
Non-Working Examples
S/N Enzyme Tween-20 Tween-80 Water Substrate (Maida)
g g g G g
1 2 0 0 4 100
2 1.5 0.2 0 3 100
3 2 0 0.2 4 100
4 1.5 0 0.2 3 100
5 1 0 0 2 100
6 2 0 0.4 4 90
7 1.5 0 0 3 90
8 1.5 0 0.4 3 90
9 1 0 0.4 2 90
10 2 0 0.4 4 90
11 0.5 0 0.5 1 90
12 6 0 0.5 12 90

Table-4
Working Examples
S/N Enzyme Tween-20 Tween-80 Water Substrate (Maida)
Unit g g g g g
1 2 0 0.5 4 100
2 2 0 0.5 4 100
3 1.5 0 0.5 3 100
4 1 0 0.5 2 100

Biscuit formulation:
The ‘dough’ was prepared from slurry with other regular biscuits ingredient listed down in Table-5 along with corn flour, maltodextrin and Aashrivaad gluten free flour.

Table-5
Biscuit Composition for 100gm
Raw Material Quantity (g)
Gluten Free Slurry: Maida/Barley/Rye 36.0
Butter 2.2
Flavor Vanilla 0.6
RPO 19.28
DMG 3.06
SMP 3.0
Sugar 22
Invert syrup 1.0
SBC 0.24
SAPP 0.12
ABC 0.35
Salt 0.35
Corn Flour 5.0
AA gluten free flour (sieved) 32.0
Maltodextrin 5.0

Example
Estimation of Gluten from the Biscuits
Sample preparation:
Four biscuits were selected randomly and powdered separately in a hygienic gluten free space. Then samples were quartered and one part of each biscuit samples were pooled and taken for the ELISA analysis.
• Briefly, 0.25g of homogenized biscuit was weighed and 2.5 ml of cocktail (patented) solution was added and mixed well by vortex.
• The same was incubated for 40 minutes at 50°C and the biscuit was let to cool down and mixed with 7.5 ml 80% aqueous ethanol.
• The vial was closed and shaken for shaker for 1 hour at room temperature and centrifuged at 2500G for 10 minutes at room temperature.
• Diluted the 80ul supernatant with 920ul (1:12.5) of dilution buffer and the same immediately used for assay. Final dilution factor is 500.
• Quantification of gluten by ELISA:
• 100 µL test diluted biscuit and standard solutions (0ppb, 5ppb, 10ppb, 20ppb, 40ppb, and 80ppb gliadin solution) were added to separate wells in duplicates and incubated for 30 minutes at room temperature.
• On completion, the liquid poured out of the wells and wells washed with 250µl diluted washing buffer. The washing step repeated two more times.
• Following the above, 100 µL of the diluted enzyme conjugate added to each well and incubated for 30 minutes at room temperature
• On completion, the liquid poured out of the wells and vigorously (three times in a row) washed as mentioned above
• Following which, 50 µL of substrate and 50 µL of chromogen were added to each well. Mixed gently by shaking the plate manually and incubate for 30 min at room temperature in dark.
• On completion of the above, added 100 µL of the stop reagent to each well. Mixed gently by shaking the plate manually, and absorbance was measured at 450 nm and read within 30 minutes.
• The absorbance value of standard and test biscuit were entered into the software RIDA SOFT Win/RIDA SOFT Win.net and the calculation is done by the use of cubic spline function. The RIDA SOFT Win indicates the results in gliadin and gluten.
, Claims:1. A pre-mix composition for reducing gluten content in gluten containing food substrates; comprising
at least one enzyme,
at least one surfactant; and
a carrier.

2. The pre-mix composition as claimed in claim 1, wherein the ratio of surfactant to enzyme is in the range from 1:2 to 1:10.

3. The pre-mix composition as claimed in claim 1, wherein the ratio of surfactant to enzyme is in the range from 1:2 to 1:4.

4. The pre-mix composition as claimed in claim 1, wherein the carrier is present in an amount range from 40 % to 70 wt % with respect to the total weight of the pre-mix.

5. The pre-mix composition as claimed in claim 1, wherein said carrier is selected from the group comprising of water, oil, organic solvents (alcohol, hexane, petroleum ether etc) or combination thereof.

6. The pre-mix composition as claimed in claim 1, wherein the carrier is water.

7. The pre-mix composition as claimed in claim 1, wherein the surfactant is selected from the group consisting of Polysorbate-20 (Tween-20), Polysorbate-60 (Tween-60), Polysorbate-80 (Tween-80), sodium stearoyl lactylate, lecithin or combinations thereof.

8. The pre-mix composition as claimed in claim 1, wherein the enzyme is obtained from microbial sources or synthetically prepared.

9. The pre-mix composition as claimed in claim 8, wherein the enzyme selected from a group consisting of papain, neutral protease, aspartic protease, cysteine protease, broad spectrum endo-protease thereof.

10. A method of treating a gluten containing food substrate with the premix composition as claimed in claims 1 to 9 to degrade/reduce the gluten content of the food substrate; said method comprising:
- contacting the premix composition with said gluten containing food substrate to obtain a slurry mixture;
- stirring the slurry mixture at a temperature ranging from 27 oC to about 45 oC at 200 rpm to 900 rpm for a period ranging from 1 to about 24 hours to obtain a slurry with gluten content below 20 ppm.

11. A food substrates treated by the premix composition as claimed in claims 1 to 9, having gluten content below 60 ppm, preferably below 40 ppm and most preferably below 20 ppm.

12. The food substrate as claimed in claim 11, wherein the substrate includes but is not limited to flours and other derivatives from wheat (such as maida, atta etc.), barley, rye.