Title of Invention	"A NOVEL SOLVENT COMPOSITION FOR PROTEIN EXTRACTION."
Abstract	A novel composition comprising Calcium chloride, Sodium Stearate and Sodium Hydroxide dissolved in water (pH of 9-11). The composition is used for obtaining isolated protein from seeds and commercially available de-oiled cake oilseeds /crop plant seeds the steps of incubation with novel composition solvent, filtration, purification, neutralization and precipitation. The precipitate is further washed, neutralized and dried to obtain the protein isolates. The specially designed solvent has a pH of 9-11 and comprises of calcium chloride, Sodium Stearate and Sodium Hydroxide dissolved in water. The calcium chloride percentage varies from 0.5-5%, Sodium Stearate 0.05-2% and sodium hydroxide to maintain the pH 9-11.

Title of Invention

"A NOVEL SOLVENT COMPOSITION FOR PROTEIN EXTRACTION."

Abstract

A novel composition comprising Calcium chloride, Sodium Stearate and Sodium Hydroxide dissolved in water (pH of 9-11). The composition is used for obtaining isolated protein from seeds and commercially available de-oiled cake oilseeds /crop plant seeds the steps of incubation with novel composition solvent, filtration, purification, neutralization and precipitation. The precipitate is further washed, neutralized and dried to obtain the protein isolates. The specially designed solvent has a pH of 9-11 and comprises of calcium chloride, Sodium Stearate and Sodium Hydroxide dissolved in water. The calcium chloride percentage varies from 0.5-5%, Sodium Stearate 0.05-2% and sodium hydroxide to maintain the pH 9-11.

Full Text	Field of the invention: The present invention relates to a novel composition for protein isolation. The solvent composition is particularly utilized for extraction for protein isolates from plants, seeds and plant parts. Background of the invention: In U.S. Pat. No. 4,889,921, Diosady et al. discloses a process for the production of protein isolates from rapeseed, including the steps of alkaline extraction and isoelectric precipitation to obtain a precipitate from which isolates are produce. So this is an acid base medium used for isolation of proteins. In the U.S. 6,905,713 the whole process comprises of salt solubilization. There are processes using either acids and bases or salts for protein extraction. Protein extraction is known from very long time, either it is concentrated proteins or isolated proteins from any oilseed crop. The steps involved for concentrated protein are dehulling of seed, extracting acid washing followed by alcoholic wash, washing of grind material and drying of material. The protein percentage of concentrated proteins is above 60%. The isolated proteins are formed by dehulling seeds, extracting oil, flaking the material, immersing in alkaline solution, heating the solution, purifying, acid precipitating, neutralizing and spray drying. So the process is acid-base neutralization process. Purification in any of the developed process is done in multi-step washing after alkaline extraction, which is very time consuming and proteins being very susceptible to microbial infections and must be processed in a short period. In contrary to the existing process of protein extraction and purification, our novel solvent medium performs the two steps/chemical reactions at a single place for extraction and purification of isolated proteins. Summary of invention The object of the present invention is to provide a novel composition for extraction of protein isolates. Another objective of the present invention is to develop a method to make a novel composition for extraction of protein isolates from plant seeds and meals. Description The instant invention provides for a novel composition for protein isolation. The novel composition is particularly useful for protein isolation from seeds of crop plants containing protein comprising the steps of incubation with specially designed solvent, filtration, purification, neutralization and precipitation. The specially designed solvent has a pH of 9-11 and comprises of calcium chloride, Sodium Stearate and Sodium Hydroxide dissolved in water. The calcium chloride percentage varies from 0.5-5%, Sodium Stearate 0.05-2% and sodium hydroxide to maintain the pH 9-11. Anti-nutritional factors associated with various crop plants like mustard seeds such as Isothiocyanates, Vinyloxazolidine-thiones, Phytic acid, Phenolic compounds can be greatly reduced, almost removed by the use of disclosed composition. In accordance with one of the embodiments of the disclosed invention the novel solvent may be used to isolate proteins from oilseeds as raw material as follows: Oilseed is cleaned, conditioned and dehulled. The dehulled seed is then cooked, flaked and oil extraction is carried out. The de-oiled cake is then hydrolyzed by applying saturated steam in the ratio of 1:20 (steam: cake) for about 30 minutes to remove any anti-nutritional elements present in it like the glucosinolates and its secondary metabolites formed during the cooking process. The material is then analyzed for the percentage of active anti-nutritional elements, if the cake is found to contain further the active anti nutritional factors, it is then baked at 60-70°C for half an hour. The baking step serves the following functions: de-activating the myrosinase enzyme and remove volatile glucosinolates and increase the Protein Dispersibility Index or Nitrogen Solubility Index PDI/NSI. The baked material is ground and immersed into an incubator containing novel solvent composition comprising a mixture of Calcium chloride (0.5%-5%), Sodium stearate (0.05%-2%) and Sodium Hydroxide (IMol/l solution), in dematerialized water (all percentages are calculated with water added in the process) pH 9-11 (maintained with Sodium Hydroxide). The ratio of raw material and water in the incubator is 1:20. The whole mixture is incubated at about 60-80°C for about 1 hour with constant stirring. The temperature variation and time period of incubation may depend upon the NSI of the parent material. The temperature may vary from 50-80°C and time from 30 minutes to l.Shrs. The material is then transferred to a series of tanks consisting of about 3 tanks with filters on the top of varying size ranging from l/2mm to 1/5 Omm. The incubated material is first filtered through a 0.5mm filter to remove the major fiber part of the material. The material is then ultra-centrifuged to remove the suspended particles from the protein solution. This material is then tested for optical analysis to analyze any further suspended particle or haziness/turbidity of the solution. The purified protein solution is then treated with 0.1-10% ethanol and 0.05-2% S.S (Sodium Stearate) and 0.01 -l%of sodium sulfite (Na2SO3). The purpose of ethanol is to make phenolic compounds soluble in water phase rather than to bound the protein part during precipitation, S.S. also helps in greatly reducing the amount of condensed tannins and phenols. Also S.S works greatly in basic phase (pH-10 and above) than in acid phase. This material is allowed to standby for 20 minutes with mild stirring and is the micro filtered with SOOmicron filters. To the final material obtained after filtration is added with 0.1-0.5% activated carbon and the mixture is kept for half an hour with constant stirring. This step is followed by neutralization to readjust the pH to isolelectric constant at pH of 3.5-4 with the help of food grade citric acid to precipitate proteins. The novel composition may also be used in the process of extracting proteins from de-oiled cake if started from commercially available meal/cake as raw material. In the incubation step the proteins become soluble in aqueous phase with maximum nitrogen dispersibility from parent material. The incubator works as a primary distributor for soluble protein water to next stage. In this process the novel solvent medium acts as a protein extractor and purifier at the same stage. Color pigments and anti-nutritional elements present in the plant seed proteins are washed away from protein body's through this novel medium and by the subsequent steps of purification. Example-1: The incubated and purified material from the above process is immersed in the prepared solvent medium containing 0.5-1% calcium chloride, 0.05-0.1% sodium stearate and 1M sodium hydroxide. The solvent is then allowed to heat for 1-li/2 hrs at 70-80°C. The immersed material is then taken out from the solvent medium, whereas the solvent medium now contains soluble proteins. The solution is then filtered and added with 0.1-0.5% ethanol, 0.05-0.1% S.S (Sodium Stearate) and 0.01 -l%of sodium sulfite (Na2SC>3). The solution is again filtered and the pH of the whole solution is maintained at 3.5 to precipitate proteins. The precipitated proteins are now washed with fresh water for 4-5times continuously and then spray dried. The purpose of ethanol is to make phenolic compounds soluble in water phase rather than to bound the protein part during precipitation, S.S. also helps in greatly reducing the amount of condensed tannins and phenols. Also S.S works greatly in basic phase (pH-10 and above) than in acid phase. This material is allowed to standby for 20 minutes with mild stirring and is the micro filtered with 500micron filters. Example -2: - The incubated and purified material from the above process is immersed in the prepared solvent medium containing 2-3% calcium chloride, 0.5-l%sodium stearate and 1M sodium hydroxide. The solvent is then allowed to heat for 1-11/2 hrs at 60-70°C. The immersed material is then taken out from the solvent medium, whereas the solvent medium now contains soluble proteins. The solution is then filtered and added with 0.1-0.5% ethanol, 0.05-0.1% S.S (Sodium Stearate) and 0.01 -0.05%of sodium sulfite (T^SOs). The solution is again filtered and the pH of the whole solution is maintained at 3.5 to precipitate proteins. The precipitated proteins are now washed with fresh water for 4-5times continuously and then spray dried. The purpose of ethanol is to make phenolic compounds soluble in water phase rather than to bound the protein part during precipitation, S.S. also helps in greatly reducing the amount of condensed tannins and phenols. Also S.S works greatly in basic phase (pH-10 and above) than in acid phase. This material is allowed to standby for 20 minutes with mild stirring and is the micro filtered with 500micron filters. Example -3: - The incubated and purified material from the above process is immersed in the prepared solvent medium containing 4-5% calcium chloride, 1.5-2% sodium stearate and IM sodium hydroxide. The solvent is then allowed to heat for 1-11/2 hrs at 80-90°C. The immersed material is then taken out from the solvent medium, whereas the solvent medium now contains soluble proteins. The solution is then filtered and added with 0.5-1% ethanol, 0.1-0.2% S.S (Sodium Stearate) and 0.01 -0.05%of sodium sulfite (NaaSOa). The solution is again filtered and the pH of the whole solution is maintained at 3.5 to precipitate proteins. The precipitated proteins are now washed with fresh water for 4-5times continuously and then spray dried. The purpose of ethanol is to make phenolic compounds soluble in water phase rather than to bound the protein part during precipitation, S.S. also helps in greatly reducing the amount of condensed tannins and phenols. Also S.S works greatly in basic phase (pH-10 and above) than in acid phase. This material is allowed to standby for 20 minutes with mild stirring and is the micro filtered with SOOmicron filters. We claim: 1. A solvent composition for protein isolation comprising calcium chloride 0.5- 5%, Sodium Stearate 0.05-2% and Sodium Hydroxide IMol/l solution dissolved in dematerialized water maintained at a pH of about 9-11. 2. The solvent composition as claimed in claim 1 wherein said solvent is allowed to heat for 1-11/2 hrs at 60-70°C, the immersed material being taken out from the solvent medium, with the solvent medium containing soluble proteins, said solution being filtered followed by addition of 0.1-0.5% ethanol, 0.05-0.1% Sodium Stearate and 0.01 -0.05%of sodium sulfite (Na2SO3). 3. The solvent composition as claimed in claim 1 wherein the protein is isolated from seeds of crop plants by incubation with said solvent followed by filtration, purification, neutralization and precipitation. 4. The solvent composition as claimed in claim 1 wherein the protein is extracted from de-oiled cake the raw material being commercially available meal/cake. 5. The solvent composition as claimed in claims 1-4 wherein the proteins become soluble in aqueous phase with maximum nitrogen dispersibility from parent material in the initial incubation step. 6. The solvent composition as claimed in claims 1-5 wherein the novel solvent medium acts as a protein extractor and purifier at the same stage. 7. The solvent composition as claimed in claims 1-6 wherein the color pigments and anti-nutritional elements present in the plant seed proteins are washed away from protein body's through this novel medium and by the subsequent steps of purification. 8. The use of the solvent composition of any of the above claims for protein extraction in a manner as herein described.

Full Text

Field of the invention:
The present invention relates to a novel composition for protein isolation. The solvent composition is particularly utilized for extraction for protein isolates from plants, seeds and plant parts.
Background of the invention:
In U.S. Pat. No. 4,889,921, Diosady et al. discloses a process for the production of protein isolates from rapeseed, including the steps of alkaline extraction and isoelectric precipitation to obtain a precipitate from which isolates are produce. So this is an acid base medium used for isolation of proteins.
In the U.S. 6,905,713 the whole process comprises of salt solubilization. There are processes using either acids and bases or salts for protein extraction.
Protein extraction is known from very long time, either it is concentrated proteins or isolated proteins from any oilseed crop. The steps involved for concentrated protein are dehulling of seed, extracting acid washing followed by alcoholic wash, washing of grind material and drying of material. The protein percentage of concentrated proteins is above 60%. The isolated proteins are formed by dehulling seeds, extracting oil, flaking the material, immersing in alkaline solution, heating the solution, purifying, acid precipitating, neutralizing and spray drying. So the process is acid-base neutralization process. Purification in any of the developed process is done in multi-step washing after alkaline extraction, which is very time consuming and proteins being very susceptible to microbial infections and must be processed in a short period.
In contrary to the existing process of protein extraction and purification, our novel solvent medium performs the two steps/chemical reactions at a single place for extraction and purification of isolated proteins.
Summary of invention
The object of the present invention is to provide a novel composition for extraction of protein isolates.
Another objective of the present invention is to develop a method to make a novel composition for extraction of protein isolates from plant seeds and meals.
Description
The instant invention provides for a novel composition for protein isolation.
The novel composition is particularly useful for protein isolation from seeds of crop plants containing protein comprising the steps of incubation with specially designed solvent, filtration, purification, neutralization and precipitation.
The specially designed solvent has a pH of 9-11 and comprises of calcium chloride, Sodium Stearate and Sodium Hydroxide dissolved in water. The calcium chloride percentage varies from 0.5-5%, Sodium Stearate 0.05-2% and sodium hydroxide to maintain the pH 9-11.
Anti-nutritional factors associated with various crop plants like mustard seeds such as Isothiocyanates, Vinyloxazolidine-thiones, Phytic acid, Phenolic compounds can be greatly reduced, almost removed by the use of disclosed composition.
In accordance with one of the embodiments of the disclosed invention the novel solvent may be used to isolate proteins from oilseeds as raw material as follows:
Oilseed is cleaned, conditioned and dehulled. The dehulled seed is then cooked, flaked and oil extraction is carried out. The de-oiled cake is then hydrolyzed by applying saturated steam in the ratio of 1:20 (steam: cake) for about 30 minutes to remove any anti-nutritional elements present in it like the glucosinolates and its secondary metabolites formed during the cooking process. The material is then analyzed for the percentage of active anti-nutritional elements, if the cake is found to contain further the active anti nutritional factors, it is then baked at 60-70°C for half an hour.
The baking step serves the following functions:
de-activating the myrosinase enzyme and remove volatile glucosinolates
and
increase the Protein Dispersibility Index or Nitrogen Solubility Index
PDI/NSI.
The baked material is ground and immersed into an incubator containing novel solvent composition comprising a mixture of Calcium chloride (0.5%-5%), Sodium stearate (0.05%-2%) and Sodium Hydroxide (IMol/l solution), in dematerialized water (all percentages are calculated with water added in the process) pH 9-11 (maintained with Sodium Hydroxide). The ratio of raw material and water in the incubator is 1:20.
The whole mixture is incubated at about 60-80°C for about 1 hour with constant stirring. The temperature variation and time period of incubation may depend upon the NSI of the parent material. The temperature may vary from 50-80°C and time from 30 minutes to l.Shrs. The material is then transferred to a series of tanks consisting of about 3 tanks with filters on the top of varying size ranging from l/2mm to 1/5 Omm.
The incubated material is first filtered through a 0.5mm filter to remove the major fiber part of the material. The material is then ultra-centrifuged to remove the suspended particles from the protein solution. This material is then tested for optical analysis to analyze any further suspended particle or haziness/turbidity of the solution. The purified protein solution is then treated with 0.1-10% ethanol and 0.05-2% S.S (Sodium Stearate) and 0.01 -l%of sodium sulfite (Na2SO3).
The purpose of ethanol is to make phenolic compounds soluble in water phase rather than to bound the protein part during precipitation, S.S. also helps in greatly reducing the amount of condensed tannins and phenols. Also S.S works greatly in basic phase (pH-10 and above) than in acid phase. This material is allowed to standby for 20 minutes with mild stirring and is the micro filtered with SOOmicron filters.
To the final material obtained after filtration is added with 0.1-0.5% activated carbon and the mixture is kept for half an hour with constant stirring. This step is followed by neutralization to readjust the pH to isolelectric constant at pH of 3.5-4 with the help of food grade citric acid to precipitate proteins.
The novel composition may also be used in the process of extracting proteins from de-oiled cake if started from commercially available meal/cake as raw material.
In the incubation step the proteins become soluble in aqueous phase with maximum nitrogen dispersibility from parent material. The incubator works as a primary distributor for soluble protein water to next stage.
In this process the novel solvent medium acts as a protein extractor and purifier at the same stage.
Color pigments and anti-nutritional elements present in the plant seed proteins are washed away from protein body's through this novel medium and by the subsequent steps of purification.
Example-1: The incubated and purified material from the above process is immersed in the prepared solvent medium containing 0.5-1% calcium chloride, 0.05-0.1% sodium stearate and 1M sodium hydroxide. The solvent is then allowed to heat for 1-li/2 hrs at 70-80°C. The immersed material is then taken out from the solvent medium, whereas the solvent medium now contains soluble proteins. The solution is then filtered and added with 0.1-0.5% ethanol, 0.05-0.1% S.S (Sodium Stearate) and 0.01 -l%of sodium sulfite (Na2SC>3). The solution is again filtered and the pH of the whole solution is maintained at 3.5 to precipitate proteins. The precipitated proteins are now washed with fresh water for 4-5times continuously and then spray dried. The purpose of ethanol is to make phenolic compounds soluble in water phase rather than to bound the protein part during precipitation, S.S. also helps in greatly reducing the amount of condensed tannins and phenols. Also S.S works greatly in basic phase (pH-10 and above) than in acid phase. This material is allowed to standby for 20 minutes with mild stirring and is the micro filtered with 500micron filters.
Example -2: - The incubated and purified material from the above process is immersed in the prepared solvent medium containing 2-3% calcium chloride, 0.5-l%sodium stearate and 1M sodium hydroxide. The solvent is then allowed to heat for 1-11/2 hrs at 60-70°C. The immersed material is then taken out from the solvent medium, whereas the solvent medium now contains soluble proteins. The solution is then filtered and added with 0.1-0.5% ethanol, 0.05-0.1% S.S (Sodium Stearate) and 0.01 -0.05%of sodium sulfite (T^SOs). The solution is again filtered and the pH of the whole solution is maintained at 3.5 to precipitate proteins. The precipitated proteins are now washed with fresh water for 4-5times continuously and then spray dried. The purpose of ethanol is to make phenolic compounds soluble in water phase rather than to bound the protein part during precipitation, S.S. also helps in greatly reducing the amount of condensed tannins and phenols. Also S.S works greatly in basic phase (pH-10 and above) than in acid phase. This material is allowed to standby for 20 minutes with mild stirring and is the micro filtered with 500micron filters.
Example -3: - The incubated and purified material from the above process is immersed in the prepared solvent medium containing 4-5% calcium chloride, 1.5-2% sodium stearate and IM sodium hydroxide. The solvent is then allowed to heat for 1-11/2 hrs at 80-90°C. The immersed material is then taken out from the solvent medium, whereas the solvent medium now contains soluble proteins. The solution is then filtered and added with 0.5-1% ethanol, 0.1-0.2% S.S (Sodium Stearate) and 0.01 -0.05%of sodium sulfite (NaaSOa). The solution is again filtered and the pH of the whole solution is maintained at 3.5 to precipitate proteins. The precipitated proteins are now washed with fresh water for 4-5times continuously and then spray dried. The purpose of ethanol is to make phenolic compounds soluble in water phase rather than to bound the protein part during precipitation, S.S. also helps in greatly reducing the amount of condensed tannins and phenols. Also S.S works greatly in basic phase (pH-10 and above) than in acid phase. This material is allowed to standby for 20 minutes with mild stirring and is the micro filtered with SOOmicron filters.

We claim:
1. A solvent composition for protein isolation comprising calcium chloride 0.5-
5%, Sodium Stearate 0.05-2% and Sodium Hydroxide IMol/l solution
dissolved in dematerialized water maintained at a pH of about 9-11.
2. The solvent composition as claimed in claim 1 wherein said solvent is allowed
to heat for 1-11/2 hrs at 60-70°C, the immersed material being taken out from
the solvent medium, with the solvent medium containing soluble proteins, said
solution being filtered followed by addition of 0.1-0.5% ethanol, 0.05-0.1%
Sodium Stearate and 0.01 -0.05%of sodium sulfite (Na2SO3).
3. The solvent composition as claimed in claim 1 wherein the protein is isolated
from seeds of crop plants by incubation with said solvent followed by
filtration, purification, neutralization and precipitation.
4. The solvent composition as claimed in claim 1 wherein the protein is extracted
from de-oiled cake the raw material being commercially available meal/cake.
5. The solvent composition as claimed in claims 1-4 wherein the proteins
become soluble in aqueous phase with maximum nitrogen dispersibility from
parent material in the initial incubation step.
6. The solvent composition as claimed in claims 1-5 wherein the novel solvent
medium acts as a protein extractor and purifier at the same stage.
7. The solvent composition as claimed in claims 1-6 wherein the color pigments
and anti-nutritional elements present in the plant seed proteins are washed
away from protein body's through this novel medium and by the subsequent
steps of purification.
8. The use of the solvent composition of any of the above claims for protein
extraction in a manner as herein described.

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Patent Number

272066

Indian Patent Application Number

629/DEL/2006

PG Journal Number

12/2016

Publication Date

18-Mar-2016

Grant Date

16-Mar-2016

Date of Filing

09-Mar-2006

Name of Patentee

MUSTARD RESEARCH AND PROMOTION CONSORTIUM

Applicant Address

307 JYOTI SHIKHAR BUILDING DISTRICT CENTRE, JANAKPURI, NEW DELHI-110058.

Inventors:

#	Inventor's Name	Inventor's Address
1	TICKOO SANJAY KUMAR	MUSTARD RESEARCH AND PROMOTION CONSORTIUM, 307 JYOTI SHIKHAR BUILDING, DISTRICT CENTRE, JANAKPURI, NEW DELHI-110058.

PCT International Classification Number

C07C, A23L 1/14

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA