Title of Invention	ENZYMATIC DEBITTERING OF POMEGRANATE JUICE
Abstract	The present invention relates to enzymatic debittering of pomegranate juice wherein a treatment of fruit juice with an optimum mixture of hydrolytic enzymes resulted in 89% reduced tannin content, 37% increased gallic acid content and 8% increased efficacy for ABTS (2,2-Azino-bis (3-ethyl benzothizdine-6 sulphonate) scavenging and a process of enzymatic debittering of pomegranate juice, comprising adding hydrolytic enzymes selected from tannase, lipase and esterase in proportion 0.5:1.2:1 (v/v) to pomegranate juice to result in a mixture, incubating the mixture at 37°C with shaking up to 120-180 min followed by heating at 50-70°C to deactivate the enzymes and to obtain the enzymatic debittering of pomegranate juice.

Title of Invention

ENZYMATIC DEBITTERING OF POMEGRANATE JUICE

Abstract

The present invention relates to enzymatic debittering of pomegranate juice wherein a treatment of fruit juice with an optimum mixture of hydrolytic enzymes resulted in 89% reduced tannin content, 37% increased gallic acid content and 8% increased efficacy for ABTS (2,2-Azino-bis (3-ethyl benzothizdine-6 sulphonate) scavenging and a process of enzymatic debittering of pomegranate juice, comprising adding hydrolytic enzymes selected from tannase, lipase and esterase in proportion 0.5:1.2:1 (v/v) to pomegranate juice to result in a mixture, incubating the mixture at 37°C with shaking up to 120-180 min followed by heating at 50-70°C to deactivate the enzymes and to obtain the enzymatic debittering of pomegranate juice.

Full Text	FIELD OF THE INVENTION: This inveniton relates to a debittered, antioxidant rich pomegranate juice. This Invention further relates to a dabittered, antioxidant rich pomegranate juice, prepared by using a hydrolyte enzyme combination. BACKGROUND OF THE INVENTION: The pomegranate (Punica granatum, Punicaceae), one of the oldest known editable fruit", affectionately known as the "Jewel of winter," has recently been acclaimed for Ms health benefits. In particular, for Ms disease-fighting antioxidant potential. There is an increased concern in the fruit juice Industry about the loss of market value of pomegranate juice due to its haziness and bitterness because of high tannin content. Adsorption techniques have been reported to achieve the role of debittering in several fruit juices. However, there is a loss of acidity, sweetness, flavor and turbidity as weR as reduced efficiency in adsorption debittering. It is known to debitter fruit juices by the use of gelatin (Vardin H & Fenercloglu H, study on the development of pomegranate juice processing technology: Clarification of pomegranate juice, Nahrung, 47 (2003) 300-303.) which precipitates the tannin by forming large cluster molecules. This method is not specific end precipitates proteins in eddition to tannin, which is undesirable. Further, gelatin is an animal protein and there are ethical constraints to the use of animals proteins in food. Therefore, it is essentiel to seek alternative modes or reagents for dibittering. Debitterlng of fruit Juices is usually carried out by add and alkali hydrolysis. This hat several drawbacks including the loss of composition and compromising the quality of tha juice through chemical reaction and removal of nutrients. Further, tha hydrolysis using acids and alkakie Is also not specific for a particular biomolecule. OBJECTS OF THE INVENTION: It is therefore an object of this Invention to provide a debittered antioxidant rich pomegranate Juice which can be obtained by a simple route. Another object of this invention is to provide a debittered antioxidant rich pomegranate Juice which is prepared without the use of harmful chemicals. Yet another object of this Invention is to provide a debittered antioxidant rich pomegranate juice which does not effect the quality of the juice These and other objects of the Invention will be apparent from the ensuing description. BRIEF DESCRIPTION OF THE INVENTION: Thus according to this invention is provided a debittered antioxidant rich pomegranate juice. According to this is further provided e process for the preparation of a debittered antioxidant rich pomegranate juice. In accordance with this invention, treatment of fruit juice with an optimum mixture of hydrorytic enzymes resulted in about 90% of tannin degradation without affecting the quality of the juice like protein content, total soluble sugar, pH, titrable acidity, organic carbon etc. A combination of enzymes is employed, i.e. tannase and lipase where lipase also Includes esterase. Tannase of activity 25 to 36 U/ml and lipase-esterase of activity of 8-14 U/ml, preferably 11 U/ml is used. To a known amount of fruit juice taken in test tube, 10-16% tannase and lipses combination, by volume of the fruit juice, is added. The proportion of tannase: lipase-esteraee employed ranges between 0.5 1.2: 1 v/v. The control test tube received only water instead of enzymes. The test tubes are then incubsted at about 37°C with gentle shaking up to 120-180 min. The test tubes are then pieced in a water bath at 60°C-70°C to deactivate the enzyme. 1ml aliquot of fruit juices is taken from each test tube at different time intervals and their tannin content is measured. Samples treated with enzymes show 32-37.9% increase in galtic acid content as compared to the untreated sample. The increase in gaic acid content is also beneficial with regard to its antioxidant potential. Treated samples show better efficacy for ABTS (2,2-Azino-bis (3-Ethyl benzothizeline-6 sulphonate) scavenging by 8% with 2µl of juice over the nontreated pomegranate juice. The invention wi now be explained in greater details with the help of the following non-limiting examples and illustrated with the help of the accompanying drawings. Examples: Chemicals All chemicals used for the following experiments wore of analytical grade. Tannate activity was determined spectrophotometrically, according to the method of buchi et al., 1961. Treatment of Juice with tannase To 10 ml of fruit juice taken in test tube, 1 ml of Tannase (36.6 U/ml) was added. The control test tube received only 1 ml of water instead of tannate. Test tubes were then incubated at 37°C with gentle shaking up to 120 min. The tett tube wat then placed in a water bath at 50°C to deactivate the enzyme. 1 ml aliquot of fruit juicet wat taken from each test tube et different time intervals and their tannin content wet meatured. The effect of tannate on the debittering of pomegranate Juicet it shown in Fig. 1. Results shown are average of five replicating experiments 1 ml of tannate (39.6 U/ml) wat uted to clarify 10 ml of fruit juice. A further increase in tannate concentration did not improve the extent of clarification Fig. 1 shows the effect of different concentrationt of tannate on tannin degradation in pomegranate fruit Juice after 2 hrs of Incubation. Values in Y-axis are represented at tannic acid equivalents. The effect of gelatin (1g/L) and tannase (1:1) combination was also tested for tannin degradation at various time intervals up to 2 hours. The gelatin and tannase combination showed a sharp decline in tannin level in fruit juices by 49% after 90 minutes of incubation (Fig. 2). Fig. 2 shows the combined effect of Tannase and gelatin (1:1) on tannin degradation. Values in Y-axis ere represented as tannic acid equivalents. Fruit Juice debittering was also tried with esterase and Npaee mixture at different time intervals due to the ability of the esterase and lipase to act on both long chain and short chain esters respectively. As esterase break all types of ester linkages it resulted in 66% tannin degradation after 2 hrs of incubation (Fig. 3 and Fig. 4). Fig 3 shows the effect of different concentration of lipase and esterase mixture on tannin degradation in pomegranate fruit juice after 2 hre of incubation. Values in Y-axis are represented as tannic acid equivalents. And Figure 4 shows the effect of Npaee and eeteraee and tannase mixture mixture on tannin degradation in pomegranate fruit juice after 2 hrs of incubation. Values in Y-axis are represented ae tannic acid equivalents. However, a mixture of tannest, Npaee and esterase of activity 11 u/ml resulted in 89% of tannin degradation with 15% of enzyme concentration (Fig 6). Treatment with this mixture also increased the gallic acid content by 37%. The treated Juice showed higher antioxidative value due to the increase concentration of gallic acid. In order to determine the change in quality due to treatment with debittering aids, the treated juice was subjected to analysis of ascorbic acid content, carbohydrate, protein, pH viscosity, titratable acidity etc. Data are presented in Table 1. Non-treated and treated samples did not show any significant variation in these aspects. However, there was an increase in total sugar content from 14.30% in non-treated juice to 15.71% in treated juice. Floribeth & Lastreto (1981) detected an increase of 20% in reducing sugar content using a combination of pectic enzymes and cellulases to clarify apple juice. Table 1: Effect of tannase on physical and chemical parameters of fruit juices Both the treated and non-treated juices were analyzed for the presence of essential elements (Table 2) by Atomic Absorption Spectroscopy. There were no significant differences in the content of iron, aluminium and zinc. But there was a meager increase in copper content in the treated juice samples. Determination of elements by Atomic absorption spectrophotometor (AAS) For determination of elements by AAS 5 ml of fruit Juice was digested with 25 ml of nitric acid and perchloric acid (9:4) for 1 hour. The digested fruit Juice was fittered through a Whatman no 42 fitter paper and the final volume was made to 100 ml. Elements content of the digested juice was determined by AAS. Table 2 Detection of Important elements in treated and non-treated fruit Juice by AAS. The enzymatically debittered juice is rich in antioxidants and there is no loss of nutritional quality of the juice upon treatment. Further, the quality of the Juice is better end there is no health hazard since the tannin levels ere lowered increased gallic acid levels are raised. WE CLAIM: 1. A debittered anti-oxidant rich pomegranate juice having 89% reduced tannin content, 37% increased gallic acid content, and an 8% increased efficacy for ABTS (2,2-Azino-bis (3-ethyl benzothiazoline-6 sulphonate) scavenging, compared to natural pomegranate juice. 2. A process for debittering anti-oxidant rich pomegranate juice, comprising adding hydrolytic enzymes selected from tannase, lipase and esterase to pomegranate juice to result in a mixture, incubating the mixture with shaking followed by heating to deactivate the enzymes, to obtain the debittered antioxidant rich pomegranate juice. 3. The process as claimed in claim 1, wherein the hydrolytic enzymes are added 10 to 16% by volume of the pomegranate juice. 4. The process as claimed in claim 2, wherein the mixture is incubated at about 37°C. 5. The process as claimed in claim 2, wherein the mixture is incubated for 120 to 180 min. 6. The process as claimed in claim 2, wherein the mixture is heated on a water bath at 50 to 70°C. 7. The process as claimed in claim 2, wherein the activity of tannase ranges from 25 to 36 U/ml. 8. The process as claimed in claim 2, wherein the activity of lipase-esterase combination ranges from 8 to 14 U/ml. The present invention relates to enzymatic debittering of pomegranate juice wherein a treatment of fruit juice with an optimum mixture of hydrolytic enzymes resulted in 89% reduced tannin content, 37% increased gallic acid content and 8% increased efficacy for ABTS (2,2-Azino-bis (3-ethyl benzothizdine-6 sulphonate) scavenging and a process of enzymatic debittering of pomegranate juice, comprising adding hydrolytic enzymes selected from tannase, lipase and esterase in proportion 0.5:1.2:1 (v/v) to pomegranate juice to result in a mixture, incubating the mixture at 37°C with shaking up to 120-180 min followed by heating at 50-70°C to deactivate the enzymes and to obtain the enzymatic debittering of pomegranate juice.

Full Text

FIELD OF THE INVENTION:
This inveniton relates to a debittered, antioxidant rich pomegranate juice.
This Invention further relates to a dabittered, antioxidant rich pomegranate juice,
prepared by using a hydrolyte enzyme combination.
BACKGROUND OF THE INVENTION:
The pomegranate (Punica granatum, Punicaceae), one of the oldest known
editable fruit", affectionately known as the "Jewel of winter," has recently been
acclaimed for Ms health benefits. In particular, for Ms disease-fighting antioxidant
potential. There is an increased concern in the fruit juice Industry about the loss
of market value of pomegranate juice due to its haziness and bitterness because
of high tannin content. Adsorption techniques have been reported to achieve the
role of debittering in several fruit juices. However, there is a loss of acidity,
sweetness, flavor and turbidity as weR as reduced efficiency in adsorption
debittering.
It is known to debitter fruit juices by the use of gelatin (Vardin H & Fenercloglu H,
study on the development of pomegranate juice processing technology:
Clarification of pomegranate juice, Nahrung, 47 (2003) 300-303.) which
precipitates the tannin by forming large cluster molecules. This method is not
specific end precipitates proteins in eddition to tannin, which is undesirable.
Further, gelatin is an animal protein and there are ethical constraints to the use of
animals proteins in food. Therefore, it is essentiel to seek alternative modes or
reagents for dibittering.
Debitterlng of fruit Juices is usually carried out by add and alkali hydrolysis. This
hat several drawbacks including the loss of composition and compromising the
quality of tha juice through chemical reaction and removal of nutrients. Further,
tha hydrolysis using acids and alkakie Is also not specific for a particular
biomolecule.
OBJECTS OF THE INVENTION:
It is therefore an object of this Invention to provide a debittered antioxidant rich
pomegranate Juice which can be obtained by a simple route.
Another object of this invention is to provide a debittered antioxidant rich
pomegranate Juice which is prepared without the use of harmful chemicals.
Yet another object of this Invention is to provide a debittered antioxidant rich
pomegranate juice which does not effect the quality of the juice
These and other objects of the Invention will be apparent from the ensuing
description.
BRIEF DESCRIPTION OF THE INVENTION:
Thus according to this invention is provided a debittered antioxidant rich
pomegranate juice.
According to this is further provided e process for the preparation of a debittered
antioxidant rich pomegranate juice.
In accordance with this invention, treatment of fruit juice with an optimum mixture
of hydrorytic enzymes resulted in about 90% of tannin degradation without
affecting the quality of the juice like protein content, total soluble sugar, pH,
titrable acidity, organic carbon etc. A combination of enzymes is employed, i.e.
tannase and lipase where lipase also Includes esterase. Tannase of activity 25 to
36 U/ml and lipase-esterase of activity of 8-14 U/ml, preferably 11 U/ml is used.
To a known amount of fruit juice taken in test tube, 10-16% tannase and lipses
combination, by volume of the fruit juice, is added. The proportion of tannase:
lipase-esteraee employed ranges between 0.5 1.2: 1 v/v. The control test tube
received only water instead of enzymes. The test tubes are then incubsted at
about 37°C with gentle shaking up to 120-180 min. The test tubes are then
pieced in a water bath at 60°C-70°C to deactivate the enzyme. 1ml aliquot of fruit
juices is taken from each test tube at different time intervals and their tannin
content is measured.
Samples treated with enzymes show 32-37.9% increase in galtic acid content as
compared to the untreated sample. The increase in gaic acid content is also
beneficial with regard to its antioxidant potential. Treated samples show better
efficacy for ABTS (2,2-Azino-bis (3-Ethyl benzothizeline-6 sulphonate)
scavenging by 8% with 2µl of juice over the nontreated pomegranate juice.
The invention wi now be explained in greater details with the help of the
following non-limiting examples and illustrated with the help of the accompanying
drawings.
Examples:
Chemicals
All chemicals used for the following experiments wore of analytical grade.
Tannate activity was determined spectrophotometrically, according to the
method of buchi et al., 1961.
Treatment of Juice with tannase
To 10 ml of fruit juice taken in test tube, 1 ml of Tannase (36.6 U/ml) was added.
The control test tube received only 1 ml of water instead of tannate. Test tubes
were then incubated at 37°C with gentle shaking up to 120 min. The tett tube
wat then placed in a water bath at 50°C to deactivate the enzyme. 1 ml aliquot of
fruit juicet wat taken from each test tube et different time intervals and their
tannin content wet meatured.
The effect of tannate on the debittering of pomegranate Juicet it shown in Fig. 1.
Results shown are average of five replicating experiments 1 ml of tannate (39.6
U/ml) wat uted to clarify 10 ml of fruit juice. A further increase in tannate
concentration did not improve the extent of clarification
Fig. 1 shows the effect of different concentrationt of tannate on tannin
degradation in pomegranate fruit Juice after 2 hrs of Incubation. Values in Y-axis
are represented at tannic acid equivalents.
The effect of gelatin (1g/L) and tannase (1:1) combination was also tested for
tannin degradation at various time intervals up to 2 hours. The gelatin and
tannase combination showed a sharp decline in tannin level in fruit juices by 49%
after 90 minutes of incubation (Fig. 2).
Fig. 2 shows the combined effect of Tannase and gelatin (1:1) on tannin
degradation. Values in Y-axis ere represented as tannic acid equivalents.
Fruit Juice debittering was also tried with esterase and Npaee mixture at different
time intervals due to the ability of the esterase and lipase to act on both long
chain and short chain esters respectively. As esterase break all types of ester
linkages it resulted in 66% tannin degradation after 2 hrs of incubation (Fig. 3 and
Fig. 4).
Fig 3 shows the effect of different concentration of lipase and esterase mixture
on tannin degradation in pomegranate fruit juice after 2 hre of incubation. Values
in Y-axis are represented as tannic acid equivalents.
And Figure 4 shows the effect of Npaee and eeteraee and tannase mixture
mixture on tannin degradation in pomegranate fruit juice after 2 hrs of incubation.
Values in Y-axis are represented ae tannic acid equivalents.
However, a mixture of tannest, Npaee and esterase of activity 11 u/ml resulted in
89% of tannin degradation with 15% of enzyme concentration (Fig 6). Treatment
with this mixture also increased the gallic acid content by 37%. The treated Juice
showed higher antioxidative value due to the increase concentration of gallic
acid.
In order to determine the change in quality due to treatment with debittering aids, the
treated juice was subjected to analysis of ascorbic acid content, carbohydrate,
protein, pH viscosity, titratable acidity etc. Data are presented in Table 1. Non-treated
and treated samples did not show any significant variation in these aspects. However,
there was an increase in total sugar content from 14.30% in non-treated juice to
15.71% in treated juice. Floribeth & Lastreto (1981) detected an increase of 20% in
reducing sugar content using a combination of pectic enzymes and cellulases to clarify
apple juice.
Table 1: Effect of tannase on physical and chemical parameters of fruit juices

Both the treated and non-treated juices were analyzed for the presence of essential
elements (Table 2) by Atomic Absorption Spectroscopy. There were no significant
differences in the content of iron, aluminium and zinc. But there was a meager
increase in copper content in the treated juice samples.
Determination of elements by Atomic absorption spectrophotometor (AAS)
For determination of elements by AAS 5 ml of fruit Juice was digested with 25 ml
of nitric acid and perchloric acid (9:4) for 1 hour. The digested fruit Juice was
fittered through a Whatman no 42 fitter paper and the final volume was made to
100 ml. Elements content of the digested juice was determined by AAS.
Table 2 Detection of Important elements in treated and non-treated fruit Juice by
AAS.
The enzymatically debittered juice is rich in antioxidants and there is no loss of
nutritional quality of the juice upon treatment. Further, the quality of the Juice is
better end there is no health hazard since the tannin levels ere lowered
increased gallic acid levels are raised.
WE CLAIM:
1. A debittered anti-oxidant rich pomegranate juice having 89% reduced
tannin content, 37% increased gallic acid content, and an 8% increased
efficacy for ABTS (2,2-Azino-bis (3-ethyl benzothiazoline-6 sulphonate)
scavenging, compared to natural pomegranate juice.
2. A process for debittering anti-oxidant rich pomegranate juice, comprising
adding hydrolytic enzymes selected from tannase, lipase and esterase to
pomegranate juice to result in a mixture, incubating the mixture with
shaking followed by heating to deactivate the enzymes, to obtain the
debittered antioxidant rich pomegranate juice.
3. The process as claimed in claim 1, wherein the hydrolytic enzymes are
added 10 to 16% by volume of the pomegranate juice.
4. The process as claimed in claim 2, wherein the mixture is incubated at
about 37°C.
5. The process as claimed in claim 2, wherein the mixture is incubated for
120 to 180 min.
6. The process as claimed in claim 2, wherein the mixture is heated on a
water bath at 50 to 70°C.
7. The process as claimed in claim 2, wherein the activity of tannase ranges
from 25 to 36 U/ml.
8. The process as claimed in claim 2, wherein the activity of lipase-esterase
combination ranges from 8 to 14 U/ml.

The present invention relates to enzymatic debittering of pomegranate juice wherein a
treatment of fruit juice with an optimum mixture of hydrolytic enzymes resulted in
89% reduced tannin content, 37% increased gallic acid content and 8% increased
efficacy for ABTS (2,2-Azino-bis (3-ethyl benzothizdine-6 sulphonate) scavenging and
a process of enzymatic debittering of pomegranate juice, comprising adding hydrolytic
enzymes selected from tannase, lipase and esterase in proportion 0.5:1.2:1 (v/v) to
pomegranate juice to result in a mixture, incubating the mixture at 37°C with shaking
up to 120-180 min followed by heating at 50-70°C to deactivate the enzymes and to
obtain the enzymatic debittering of pomegranate juice.

Documents:

00613-kol-2005-description provisional.pdf

00613-kol-2005-form 1.pdf

00613-kol-2005-form 2.pdf

00613-kol-2005-form 3.pdf

613-kol -2005-granted-abstract.pdf

613-kol -2005-granted-claims.pdf

613-kol -2005-granted-correspondence.pdf

613-kol -2005-granted-description (complete).pdf

613-kol -2005-granted-drawings.pdf

613-kol -2005-granted-examination report.pdf

613-kol -2005-granted-form 1.pdf

613-kol -2005-granted-form 18.pdf

613-kol -2005-granted-form 2.pdf

613-kol -2005-granted-form 3.pdf

613-kol -2005-granted-form 5.pdf

613-kol -2005-granted-gpa.pdf

613-kol -2005-granted-reply to examination report.pdf

613-kol -2005-granted-specification.pdf

613-kol-2005-abstract.pdf

613-kol-2005-claims.pdf

613-kol-2005-correspondence.pdf

613-kol-2005-description (complete).pdf

613-kol-2005-drawings.pdf

613-kol-2005-examination report.pdf

613-kol-2005-form 1.pdf

613-kol-2005-form 18.pdf

613-kol-2005-form 2.pdf

613-kol-2005-form 3.pdf

613-kol-2005-form 5.pdf

613-kol-2005-gpa.pdf

613-kol-2005-reply to examination report.pdf

613-kol-2005-specification.pdf

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Patent Number

235908

Indian Patent Application Number

613/KOL/2005

PG Journal Number

36/2009

Publication Date

04-Sep-2009

Grant Date

03-Sep-2009

Date of Filing

13-Jul-2005

Name of Patentee

INDIAN INSTITUTE OF TECHNOLOGY

Applicant Address

KHARAGPUR

Inventors:

#	Inventor's Name	Inventor's Address
1	RINTU BANERJEE	C/O. INDIAN INSTITUTE OF TECHONOLOGY KHARAGPUR 721 302
2	PARAMITA MAHAPATRA	C/O. INDIAN INSTITUTE OF TECHONOLOGY KHARAGPUR 721 302
3	ANNAPURNA KUMARI	C/O. INDIAN INSTITUTE OF TECHONOLOGY KHARAGPUR 721 302
4	SURYAMANI ROUT	C/O. INDIAN INSTITUTE OF TECHONOLOGY KHARAGPUR 721 302

PCT International Classification Number

A23L2

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA