Title of Invention	"A PROCESS FOR EXTRACTION AND PURIFICATION OF MUSTARD PROTEIN ISOLATES FROM DE-OILED MUSTARD CAKE AND SYSTEM THEREOF"
Abstract	A process and system for obtaining isolated protein from seeds and commercially available de-oiled cake of mustard comprising the steps of incubation with specially designed 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.

Title of Invention

"A PROCESS FOR EXTRACTION AND PURIFICATION OF MUSTARD PROTEIN ISOLATES FROM DE-OILED MUSTARD CAKE AND SYSTEM THEREOF"

Abstract

A process and system for obtaining isolated protein from seeds and commercially available de-oiled cake of mustard comprising the steps of incubation with specially designed 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.

Full Text	Field of the invention: The present invention relates to a new technology for extraction and purification of proteins from oil seeds and de-oiled cake, more particularly the invention relates to extraction of protein from mustard seed and de-oiled cake. Background of the invention: There are many sources of Proteins such as the Caseins, Whey Proteins, Muscle Proteins, Soy Proteins, Cereal Proteins, Seaweed Proteins and Proteins from Oil-Producing Plants. Proteins isolated from oil-producing plants have not yet been commercially exploited to its complete potential. Very few protein extraction processes for protein isolation of these plants are known, as the anti-nutritional elements are difficult to be separated from the proteins, especially mustard or rapeseed. Diosady et al. (In US4889921) discloses a process for the production of protein isolates from mustard seeds. The process comprises the steps of alkaline extraction and isoelectric precipitation to obtain a precipitate from which a first product stream of protein is recovered. The depleted solution from the precipitation stage is subjected to ultrafiltration followed by diafiltration and drying to obtain a second product stream of recovered protein. These two protein isolates are produced with a combined protein recovery of over 70% of the protein present in the seed. Both products are of high protein content (>90%), essentially free of glucosinolates ( Membrane processes are used to concentrate and purify the protein isolates In the US4889921 patent. These processes are effective at removing the glucosinolates and the phytates, as they are relatively small and pass through the pores of the membrane. However, the relatively large phenolic-protein complexes tend to be rejected by the membrane, and thus remain behind in the retentate along with the protein isolates. The US6905713 process comprises of salt solubilization for making phenolic compound complexes free followed by ultra-filtration and dia-filtration to get isolated rapeseed proteins. However the process is not economical for a large scale pilot plant wherein a simple process and system is desirable. In order to overcome the drawbacks of the prior art the instant invention introduces for a new process for extraction and purification of protein from mustard. Summary of invention The object of the present invention is to provide a method and system for extraction and purification of protein isolates of oil-seed wherein a newly designed solvent medium is used for protein isolation. The objective of the present invention is to develop an economic process isolation of proteins from mustard seed and de-oiled cake. Further, the invention provides a method and system to obtain protein isolates of more than 90% purity from mustard. Brief Description Of The Accompanying Drawings and Tables The Figure 1 describes the steps of the process of extraction of protein isolates from mustard. Table-1 provides data on Anti-nutritional factors of parent material, de-oiled cake and isolated mustard protein Table-2 provides data on composition of parent material, de-oiled mustard cake and mustard protein isolate. Detailed Description Other objects and aspects of the invention will become apparent from the following description and the embodiments with reference to the accompanying drawings, which is set forth hereinafter. The embodiments of the present invention can be modified variously. Thus, the scope of the present invention should be construed not limited to the description and embodiments provided herein. The embodiments are provided to better explain the present invention to those of ordinary skill in the art. The instant invention provides for a process and system for obtaining isolated protein from seeds as well as de-oiled cake of mustard comprising the steps of incubation with specially designed 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 flow chart shown in figure 1 describes the various steps of the process in a schematic manner. Anti-nutritional factors such as Isothiocyanates, Vinyloxazolidine-thiones, Phytic acid, Phenolic compounds greatly reduced, almost removed by the disclosed process. The Anti-nutritional factors of parent material and the protein isolate obtained have been summarized in Table 1. (Table Removed) In accordance with one of the embodiments of the disclosed invention the process to isolate proteins from mustard using mustard seed as raw material may be carried out in the following manner: Mustard seed is cleaned, conditioned and dehulled. The dehulled Mustard 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 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 designed solvent medium which is a mixture of Calcium chloride (0.5%), Sodium stearate (0.05%) and Sodium Hydroxide (1Mol/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 mustard de-oiled cake 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 1 .5hrs. 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 1/2mm to 1/50mm. The incubated material is first transferred in tank-1, which has 0.5mm filter to remove the major fiber part of the material. The material obtained from first tank is then ultra-centrifuged to remove the suspended particles from the protein solution. This material was then transferred to tank-2. at this stage the solution is then tested for optical analysis to analyze any further suspended particle or haziness/turbidity of the solution. If any kind of problem is found at this stage using the varying size of filters as mentioned above further purifies it. The transparency of the solution is very important to prevent loss of low molecular weight proteins. The purified protein solution is then transferred to tank-3 where it is treated with 0.1% ethanol and 0.05% S.S (Sodium Stearate) and 0.01% of sodium sulfite 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 gratly 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. To the final material obtained after filtration is added with 0.1% 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. This process is conducted in a tank graduated with automatic pH-meter to constantly monitor the pH of the solution. At this stage a second process for precipitation of isolated proteins is carried out by thermal precipitation/coagulation by injecting steam in the water-protein phase by raising the temperature to about 90 ±5°C and then cooling and centrifugation to precipitate proteins. Before final washing of precipitated proteins, S.S. is added at 0.01% on water volume basis in first 2-3 washes to remove further bound phenolics. The precipitated proteins are then washed with fresh dematerialized water almost 6-0 times to remove all the anti nutritional compounds from the precipitated proteins. Na2S03 is used in this step to prevent darkening of isolated mustard proteins as an anti-oxidant, particularly against phenolic compounds. Extra water is removed from the precipitate to maintain the water and isolate ratio of about 1:20. The wet protein is neutralized preferably with citric acid or NaoH to get isolated soluble mustard protein. The wet protein is vacuum evaporated to concentrate the proteins upto 20-25% and then dried. The final mustard soluble protein isolate was obtained with a purity of more than 90%. The process of extracting proteins from mustard using de-oiled mustard cake as raw material may be carried out in the following manner: The process can start from the locally available mustard de-oiled cake from any solvent extracted unit. Grinding of de-oiled mustard cake and activation of myrosinase under hydrolysis prepares the mustard de-oiled cake for the process. The activation of myrosinase for hydrolysis is done by saturated steam injected in the chamber at 1:20 ratio (Steam: cake). The ground material of de-oiled mustard cake is immersed in acidic water medium in the newly designed incubator with constant stirring maintaining the pH at 4.5, water and de-oiled cake ratio 1:10 to prevent loss of proteins, the pH is maintained with Citric acid (2mol/l). The temperature of the medium is 50-60°C for 20-25 minutes. This medium also contained 0.5% activated carbon, 0.1% ethanol and 0.01% sodium Stearate (S.S). This step is basically cake-conditioning for protein extraction. The material is now analyzed for the percentage of active anti-nutritional elements, if the cake has found to contain further the active anti nutritional factors, it is then baked at 60°C for half an hour. This baking helps in two ways one is the de-activating the myrosinase enzyme and remove volatile glucosinolates and the second is to increase the Protein Dispersibility Index or Nitrogen Solubility Index PDI/NSI. Now the baked material is ground and immersed into a newly developed incubator. The incubator contains a mixture of Calcium chloride (0.5%), Sodium stearate (0.05%) and Sodium Hydroxide (1Mol/l solution) to maintain the pH of 9-11 in dematerialized water (all percentages are calculated with water added in the process). The Mustard de-oiled cake and water ratio in the incubator is 1:20. The whole mixture is then incubated in the incubator (for 30-60 minutes) at 50°-80°C along with the designed solvent medium and all steps are carried out similar to those describe before for the process of extraction of proteins from mustard seed. The quantity of de-oiled mustard cake used and the volume of water utilized for the process defines the mass balance. In this process the NDI is increased to increase the protein production. The Chemical Composition of the Parent material and isolates obtained from it are listed in Table 2. The ± variations are dependent on standard process described. The Mass Balance may be represented as: Amount of protein present in mustard de-oiled cake Processing loss Protein purity (Table Removed) The system for carrying out the process of isolation of protein from mustard seed consists of an incubator tank with auto temperature controller and stirrer, a branched pipeline to different related equipments, an industrial centrifuge, a series of purification and filtration tanks, specially designed filters, automatic vacuum evaporators and driers. In one embodiment of the invention the incubator has a thermostat and an automatic temperature controller to maintain the temperature in the range of from 20°C to 100°C. The incubator preferable comprises of food grade stainless steel body, a stirrer, digital activity monitor, an inlet and an out let with motor control. The ultra-centrifuge of the system runs on a spindle base with stainless steel mesh of varying size (changeable) with a broad inlet and a narrow outlet with changeable rotor of varying size. The series of purification and filtration tanks may be made of food grade stainless steel fitted with level gauge and multi outlet which is best suited for water decantation. The dehumidifier is preferably fitted with digital controllers and monitors to adjust the degree of heat or vacuum. In another embodiment of the invention the drier may be a spray drier or hot air drier specially designed for drying wet isolated proteins obtained at the end of the process. The drier is fitted with digital controllers and monitors with varying inlet and out temperature and may also have a cyclone separator. 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. Centrifugation step separates the protein from the fiber and purifies the protein water. In the next step a series of purification and filtration tanks are used to purify and isolate protein from aqueous phase in precipitate form. The specially designed filters are then utilized to remove any unwanted material from the aqueous phase. The vacuum evaporator concentrates the isolated proteins by decreasing the moisture level and the drier further reduces the moisture level of the isolated proteins to 5-6%. It is one of the preferred embodiments to maintain the temperature of the incubator at 70°C. It is another preferred embodiment to carry out the incubation at pH 11 for better recovery of the proteins. At 70°C and 11 pH in the incubator the extraction recovery was found to have increased. Also the precipitated proteins settled well during washings. However, Temperature of the incubation kept stable at 50°C has a negative effect with the recovery of proteins reducing by 10% and also tan-brown color of the protein is obtained which is undesirable. In one of the embodiments activated carbon is used along with sparging of live steam in the system. The use of the activated carbon and live-steam sparging prevents the development of off-flavor and dark color, and also prevents loss of low molecular weight protein. While the present invention has been described with respect to certain preferred embodiments, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention as defined in the claims. We claim: 1. A process for extraction of protein isolates from mustard comprising: a) conditioning de-oiled mustard cake obtained from mustard seed, b) Incubating the conditioned de-oiled cake with a solvent in the ratio of 1:20, at 60-80°C for atleast 45 minutes with constant stirring, c) passing the incubated material through a series of filtration tanks to obtain First Extract and pulp, d) ultra-centrifugation of First Extract to remove fibers and obtain Second Extract, e) washing and filtrating the pulp to obtain Third Extract, f) treating Second and Third Extract with 0.1% ethanol and 0.05% SS (Sodium Stearate) and 0.01% of sodium sulfite (Na2SO3), for 20 minutes with mild stirring followed by micro filteration to obtain mustard protein, g) adjusting the pH of mustard protein to isoelectric constant. h) precipitation of said protein followed by cooling and centrifugation, i) washing the precipitated proteins with 0.2% activated carbon and 0.01% Sodium Stearate followed by 6-10 times washing in fresh dematerialized water to obtain wet protein, j) neutralization of wet protein with citric acid/NaOH, k) concentration of wet protein by vacuum evaporation, and I) drying to obtain the protein isolates. 2. The process for extraction of mustard protein isolates as claimed in claim 1 wherein said solvent comprises of Calcium chloride (0.5%), Sodium Stearate (0.05%) and Sodium Hydroxide (1Mol/l solution) in water at pH of 9-11. 3. A process for extraction of mustard protein isolates as claimed in claim 1 wherein said de-oiled mustard cake is obtained from the mustard seed by conditioning and dehulling of mustard seed followed by extraction of oil. 4. A process for extraction of protein isolates as claimed in claim 1 or 3 wherein said conditioning of de-oiled mustard cake is by applying saturated steam for atleast 20 minutes in the ratio of 1:20. 5. A process for extraction of protein isolates as claimed in claim 1 wherein the de-oiled mustard cake is conditioned by a) grinding of de-oiled cake and activation of myrosinase under hydrolysis, b) incubating the above treated de-oiled cake in acidic medium (pH 4.5) in the ration 1:10 with constant stirring at 50-60°C for 20-25 minutes wherein the acidic medium comprises of Citric acid, 0.5% activated carbon, 0.1% ethanol and 0.01% sodium sulfite (^SOs), and c) washing atleast once the acid-incubated de-oiled cake followed by centrifugation. 6. A process for extraction of protein isolates as claimed in claim 1 wherein said adjustment of the pH to isoelectric constant of mustard protein is by neutralization using weak acid/base. 7. A process for extraction of protein isolates as claimed in claim 1 wherein the precipitation of protein is by injecting steam / citric acid in the water- protein phase at about 90 ±5°C. 8. A system for extracting protein from mustard seed or de-oiled cake comprising: a) an incubator for extracting protein in the aqueous phase, b) a plurality of purifications and filtration tanks, c) a plurality of connecting pipelines, d) at least one centrifuge, e) a plurality of filters specially designed to suit the isolation of pure proteins with minimum losses, f) atleast one neutralization unit, g) at least one vacuum evaporator and h) at least one drier. Wherein said incubator comprises of automatic temperature controller to set the temperature from 20°C to 100°C, a thermostat, food grade stainless steel body, a stirrer, digital activity monitor, an inlet and an out let with motor control. 9. A system for extracting protein from mustard seed or de-oiled cake as claimed in claim 1 wherein said centrifuge is an ultra-centrifuge running on a spindle base with stainless steel mesh of varying size (changeable) with a broad inlet and a narrow outlet with changeable rotor of varying size. 10. A system for extracting protein from mustard seed or de-oiled cake as claimed in claim 1 wherein said plurality of purification and filtration tanks are arranged in a series and are made of food grade stainless steel fitted with level gauge and multi outlet best suited for liquid decantation. 11. A system for extracting protein from mustard seed or de-oiled cake as claimed in claim 1 wherein said vacuum evaporator works under desired vacuum conditions and has a dehumidifier fitted with digital controllers and monitors to adjust the degree of heat or vacuum. 12. A system for extracting protein from mustard seed or de-oiled cake as claimed in claim 1 wherein said drier is either a spray drier or a hot air drier fitted with digital controllers and monitors with varying inlet and out temperature and having cyclone separator. 13. A process for extraction of protein isolates from mustard seed or de-oiled cake substantially as herein described with reference to accompanying drawings. 14. A process for extraction of protein isolates from de-oiled mustard cake of any source substantially as herein described with reference to accompanying drawings. 15. A system for extracting protein from mustard seed or de-oiled cake substantially as herein described with reference to accompanying drawings.

Full Text

Field of the invention:
The present invention relates to a new technology for extraction and purification of proteins from oil seeds and de-oiled cake, more particularly the invention relates to extraction of protein from mustard seed and de-oiled cake.
Background of the invention:
There are many sources of Proteins such as the Caseins, Whey Proteins, Muscle Proteins, Soy Proteins, Cereal Proteins, Seaweed Proteins and Proteins from Oil-Producing Plants.
Proteins isolated from oil-producing plants have not yet been commercially exploited to its complete potential. Very few protein extraction processes for protein isolation of these plants are known, as the anti-nutritional elements are difficult to be separated from the proteins, especially mustard or rapeseed.
Diosady et al. (In US4889921) discloses a process for the production of protein isolates from mustard seeds. The process comprises the steps of alkaline extraction and isoelectric precipitation to obtain a precipitate from which a first product stream of protein is recovered. The depleted solution from the precipitation stage is subjected to ultrafiltration followed by diafiltration and drying to obtain a second product stream of recovered protein. These two protein isolates are produced with a combined protein recovery of over 70% of the protein present in the seed. Both products are of high protein content (>90%), essentially free of glucosinolates ( Membrane processes are used to concentrate and purify the protein isolates In the US4889921 patent. These processes are effective at removing the glucosinolates and the phytates, as they are relatively small and pass through the pores of the membrane. However, the relatively large phenolic-protein complexes tend to be rejected by the membrane, and thus remain behind in the retentate along with the protein isolates.
The US6905713 process comprises of salt solubilization for making phenolic compound complexes free followed by ultra-filtration and dia-filtration to get isolated rapeseed proteins. However the process is not economical for a large scale pilot plant wherein a simple process and system is desirable.
In order to overcome the drawbacks of the prior art the instant invention introduces for a new process for extraction and purification of protein from mustard.
Summary of invention
The object of the present invention is to provide a method and system for extraction and purification of protein isolates of oil-seed wherein a newly designed solvent medium is used for protein isolation.
The objective of the present invention is to develop an economic process isolation of proteins from mustard seed and de-oiled cake.
Further, the invention provides a method and system to obtain protein isolates of more than 90% purity from mustard.
Brief Description Of The Accompanying Drawings and Tables
The Figure 1 describes the steps of the process of extraction of protein isolates from mustard.
Table-1 provides data on Anti-nutritional factors of parent material, de-oiled cake and isolated mustard protein
Table-2 provides data on composition of parent material, de-oiled mustard cake and mustard protein isolate.
Detailed Description
Other objects and aspects of the invention will become apparent from the following description and the embodiments with reference to the accompanying drawings, which is set forth hereinafter. The embodiments of the present invention can be modified variously. Thus, the scope of the present invention should be construed not limited to the description and embodiments provided herein. The embodiments are provided to better explain the present invention to those of ordinary skill in the art.
The instant invention provides for a process and system for obtaining isolated protein from seeds as well as de-oiled cake of mustard comprising the steps of incubation with specially designed 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 flow chart shown in figure 1 describes the various steps of the process in a schematic manner.
Anti-nutritional factors such as Isothiocyanates, Vinyloxazolidine-thiones, Phytic acid, Phenolic compounds greatly reduced, almost removed by the disclosed process. The Anti-nutritional factors of parent material and the protein isolate obtained have been summarized in Table 1.
(Table Removed)
In accordance with one of the embodiments of the disclosed invention the process to isolate proteins from mustard using mustard seed as raw material may be carried out in the following manner:
Mustard seed is cleaned, conditioned and dehulled. The dehulled Mustard 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 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 designed solvent medium which is a mixture of Calcium chloride (0.5%), Sodium stearate (0.05%) and Sodium Hydroxide (1Mol/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 mustard de-oiled cake 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 1 .5hrs. 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 1/2mm to 1/50mm.
The incubated material is first transferred in tank-1, which has 0.5mm filter to remove the major fiber part of the material. The material obtained from first tank is then ultra-centrifuged to remove the suspended particles from the protein solution. This material was then transferred to tank-2. at this stage the solution is then tested for optical analysis to analyze any further suspended particle or haziness/turbidity of the solution. If any kind of problem is found at this stage using the varying size of filters as mentioned above further purifies it. The transparency of the solution is very important to prevent loss of low molecular weight proteins. The purified protein solution is then transferred to tank-3 where it is treated with 0.1% ethanol and 0.05% S.S (Sodium Stearate) and 0.01% of sodium sulfite
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 gratly 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.
To the final material obtained after filtration is added with 0.1% 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. This process is conducted in a tank graduated with automatic pH-meter to constantly monitor the pH of the solution.
At this stage a second process for precipitation of isolated proteins is carried out by thermal precipitation/coagulation by injecting steam in the water-protein phase by raising the temperature to about 90 ±5°C and then cooling and centrifugation to precipitate proteins.
Before final washing of precipitated proteins, S.S. is added at 0.01% on water volume basis in first 2-3 washes to remove further bound phenolics. The precipitated proteins are then washed with fresh dematerialized water almost 6-0 times to remove all the anti nutritional compounds from the precipitated
proteins. Na2S03 is used in this step to prevent darkening of isolated mustard proteins as an anti-oxidant, particularly against phenolic compounds.
Extra water is removed from the precipitate to maintain the water and isolate ratio of about 1:20. The wet protein is neutralized preferably with citric acid or NaoH to get isolated soluble mustard protein. The wet protein is vacuum evaporated to concentrate the proteins upto 20-25% and then dried.
The final mustard soluble protein isolate was obtained with a purity of more than 90%.
The process of extracting proteins from mustard using de-oiled mustard cake as raw material may be carried out in the following manner:
The process can start from the locally available mustard de-oiled cake from any solvent extracted unit. Grinding of de-oiled mustard cake and activation of myrosinase under hydrolysis prepares the mustard de-oiled cake for the process. The activation of myrosinase for hydrolysis is done by saturated steam injected in the chamber at 1:20 ratio (Steam: cake).
The ground material of de-oiled mustard cake is immersed in acidic water medium in the newly designed incubator with constant stirring maintaining the pH at 4.5, water and de-oiled cake ratio 1:10 to prevent loss of proteins, the pH is maintained with Citric acid (2mol/l). The temperature of the medium is 50-60°C for 20-25 minutes. This medium also contained 0.5% activated carbon, 0.1% ethanol and 0.01% sodium Stearate (S.S). This step is basically cake-conditioning for protein extraction.
The material is now analyzed for the percentage of active anti-nutritional elements, if the cake has found to contain further the active anti nutritional factors, it is then baked at 60°C for half an hour. This baking helps in two ways one is the de-activating the myrosinase enzyme and remove volatile glucosinolates and the second is to increase the Protein Dispersibility Index or Nitrogen Solubility Index PDI/NSI. Now the baked material is ground and immersed into a newly developed incubator. The incubator contains a mixture of Calcium chloride (0.5%), Sodium stearate (0.05%) and Sodium Hydroxide (1Mol/l solution) to maintain the pH of 9-11 in dematerialized water (all percentages are calculated with water added in the process). The Mustard de-oiled cake and water ratio in the incubator is 1:20.
The whole mixture is then incubated in the incubator (for 30-60 minutes) at 50°-80°C along with the designed solvent medium and all steps are carried out similar to those describe before for the process of extraction of proteins from mustard seed.
The quantity of de-oiled mustard cake used and the volume of water utilized for the process defines the mass balance. In this process the NDI is increased to increase the protein production.
The Chemical Composition of the Parent material and isolates obtained from it are listed in Table 2.
The ± variations are dependent on standard process described. The Mass Balance may be represented as:
Amount of protein present in mustard de-oiled cake Processing loss Protein purity
(Table Removed)
The system for carrying out the process of isolation of protein from mustard seed consists of an incubator tank with auto temperature controller and stirrer, a branched pipeline to different related equipments, an industrial centrifuge, a series of purification and filtration tanks, specially designed filters, automatic vacuum evaporators and driers.
In one embodiment of the invention the incubator has a thermostat and an automatic temperature controller to maintain the temperature in the range of from 20°C to 100°C. The incubator preferable comprises of food grade stainless steel body, a stirrer, digital activity monitor, an inlet and an out let with motor control. The ultra-centrifuge of the system runs on a spindle base with stainless steel mesh of varying size (changeable) with a broad inlet and a narrow outlet with changeable rotor of varying size. The series of purification and filtration tanks may be made of food grade stainless steel fitted with level gauge and multi outlet which is best suited for water decantation. The dehumidifier is preferably fitted with digital controllers and monitors to adjust the degree of heat or vacuum. In another embodiment of the invention the drier may be a spray drier or hot air drier specially designed for drying wet isolated proteins obtained at the end of the process. The drier is fitted with digital controllers and monitors with varying inlet and out temperature and may also have a cyclone separator.
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.
Centrifugation step separates the protein from the fiber and purifies the protein water. In the next step a series of purification and filtration tanks are used to purify and isolate protein from aqueous phase in precipitate form. The specially designed filters are then utilized to remove any unwanted material from the aqueous phase. The vacuum evaporator concentrates the isolated proteins by decreasing the moisture level and the drier further reduces the moisture level of the isolated proteins to 5-6%.
It is one of the preferred embodiments to maintain the temperature of the incubator at 70°C. It is another preferred embodiment to carry out the incubation at pH 11 for better recovery of the proteins.
At 70°C and 11 pH in the incubator the extraction recovery was found to have increased. Also the precipitated proteins settled well during washings.
However, Temperature of the incubation kept stable at 50°C has a negative effect with the recovery of proteins reducing by 10% and also tan-brown color of the protein is obtained which is undesirable.
In one of the embodiments activated carbon is used along with sparging of live steam in the system. The use of the activated carbon and live-steam sparging prevents the development of off-flavor and dark color, and also prevents loss of low molecular weight protein.
While the present invention has been described with respect to certain preferred embodiments, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention as defined in the claims.

We claim:
1. A process for extraction of protein isolates from mustard comprising:
a) conditioning de-oiled mustard cake obtained from mustard seed,
b) Incubating the conditioned de-oiled cake with a solvent in the ratio
of 1:20, at 60-80°C for atleast 45 minutes with constant stirring,
c) passing the incubated material through a series of filtration tanks to
obtain First Extract and pulp,
d) ultra-centrifugation of First Extract to remove fibers and obtain
Second Extract,
e) washing and filtrating the pulp to obtain Third Extract,
f) treating Second and Third Extract with 0.1% ethanol and 0.05% SS
(Sodium Stearate) and 0.01% of sodium sulfite (Na2SO3), for 20
minutes with mild stirring followed by micro filteration to obtain
mustard protein,
g) adjusting the pH of mustard protein to isoelectric constant.
h) precipitation of said protein followed by cooling and centrifugation,
i) washing the precipitated proteins with 0.2% activated carbon and 0.01% Sodium Stearate followed by 6-10 times washing in fresh dematerialized water to obtain wet protein,
j) neutralization of wet protein with citric acid/NaOH,
k) concentration of wet protein by vacuum evaporation, and
I) drying to obtain the protein isolates.
2. The process for extraction of mustard protein isolates as claimed in claim 1
wherein said solvent comprises of Calcium chloride (0.5%), Sodium Stearate
(0.05%) and Sodium Hydroxide (1Mol/l solution) in water at pH of 9-11.
3. A process for extraction of mustard protein isolates as claimed in claim 1
wherein said de-oiled mustard cake is obtained from the mustard seed by
conditioning and dehulling of mustard seed followed by extraction of oil.
4. A process for extraction of protein isolates as claimed in claim 1 or 3
wherein said conditioning of de-oiled mustard cake is by applying saturated
steam for atleast 20 minutes in the ratio of 1:20.
5. A process for extraction of protein isolates as claimed in claim 1 wherein
the de-oiled mustard cake is conditioned by

a) grinding of de-oiled cake and activation of myrosinase under
hydrolysis,
b) incubating the above treated de-oiled cake in acidic medium (pH
4.5) in the ration 1:10 with constant stirring at 50-60°C for 20-25
minutes wherein the acidic medium comprises of Citric acid, 0.5%
activated carbon, 0.1% ethanol and 0.01% sodium sulfite (^SOs),
and
c) washing atleast once the acid-incubated de-oiled cake followed by
centrifugation.

6. A process for extraction of protein isolates as claimed in claim 1 wherein
said adjustment of the pH to isoelectric constant of mustard protein is by
neutralization using weak acid/base.
7. A process for extraction of protein isolates as claimed in claim 1 wherein
the precipitation of protein is by injecting steam / citric acid in the water-
protein phase at about 90 ±5°C.
8. A system for extracting protein from mustard seed or de-oiled cake
comprising:

a) an incubator for extracting protein in the aqueous phase,
b) a plurality of purifications and filtration tanks,
c) a plurality of connecting pipelines,
d) at least one centrifuge,
e) a plurality of filters specially designed to suit the isolation of pure
proteins with minimum losses,
f) atleast one neutralization unit,
g) at least one vacuum evaporator and
h) at least one drier.
Wherein said incubator comprises of automatic temperature controller to set the temperature from 20°C to 100°C, a thermostat, food grade stainless steel body, a stirrer, digital activity monitor, an inlet and an out let with motor control.
9. A system for extracting protein from mustard seed or de-oiled cake as
claimed in claim 1 wherein said centrifuge is an ultra-centrifuge running on a spindle base with stainless steel mesh of varying size (changeable) with a broad inlet and a narrow outlet with changeable rotor of varying size.
10. A system for extracting protein from mustard seed or de-oiled cake as
claimed in claim 1 wherein said plurality of purification and filtration tanks are
arranged in a series and are made of food grade stainless steel fitted with
level gauge and multi outlet best suited for liquid decantation.
11. A system for extracting protein from mustard seed or de-oiled cake as
claimed in claim 1 wherein said vacuum evaporator works under desired
vacuum conditions and has a dehumidifier fitted with digital controllers and
monitors to adjust the degree of heat or vacuum.
12. A system for extracting protein from mustard seed or de-oiled cake as
claimed in claim 1 wherein said drier is either a spray drier or a hot air drier
fitted with digital controllers and monitors with varying inlet and out
temperature and having cyclone separator.
13. A process for extraction of protein isolates from mustard seed or de-oiled
cake substantially as herein described with reference to accompanying
drawings.
14. A process for extraction of protein isolates from de-oiled mustard cake of
any source substantially as herein described with reference to accompanying
drawings.
15. A system for extracting protein from mustard seed or de-oiled cake
substantially as herein described with reference to accompanying drawings.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=ILp9bd4XkOY48ffIXRzlRA==&loc=+mN2fYxnTC4l0fUd8W4CAA==

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Patent Number

269001

Indian Patent Application Number

694/DEL/2006

PG Journal Number

40/2015

Publication Date

02-Oct-2015

Grant Date

28-Sep-2015

Date of Filing

14-Mar-2006

Name of Patentee

MUSTARD RESEARCH AND PROMOTION CONSORTIUM

Applicant Address

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

Inventors:

#	Inventor's Name	Inventor's Address
1	TICKOO, SANJAY KUMAR	307 JYOTI SHIKHAR BUILDING DISTRICT CENTRE, JANAKPURI, NEW DELHI-110058, INDIA

PCT International Classification Number

A23J 1/14

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA