Field of the Invention

The present invention related to an improved process for the separation of Ovotransferrinfromegg white.

Background of the Invention

Ovotransferrin is one of the major egg white proteins that have antimicrobial activity as well as iron binding capability. Ovotransferrin a monomeric glycoprotein consisting of 686 amino acids and 15 disulfide bonds. It was first characterized by Schade and Caroline in 1944 and was called Conalbuminutes. It was renamed as ovotransferrin after the finding that it can bind iron (Williams, 1968) and has the same amin o acid sequence as the trans-ferrin in human serum. The molecular weight of ovotransferrin has 76 kDa and an isoelectric point of 6.1. One ovotransferrin molecule can bind 2 iron molecules and transports iron into the body. Ovotransferrin is known to have a strong antimicrobial activity, and thus, can be used to improve the safety of foods. Peptides derived from ovotransferrin also have an ability to control microorganisms. Therefore, both ovotransferrin and its peptides can be used as antimicrobial agents in foods. Ovotransferrin is found in 2 main forms: apo- (iron free) and the holo- (iron bound). The chemical and physical properties of these 2 forms of Ovotransferrin differ significantly. Apo form (iron free) is colourless, whereas holo-form (iron bound) has a salmon pink colour. The holo-form is more resistant to chemical and physical conditions than Apo form. Iron ion (Fe3+) can be easily attached to ovotransferrin at pH >7.0, but is released at pH<4.5.
Literature reported the processes for separation of Ovotransferrin has been disclosed inK Y K.o et als’s 2008 Poultry Science 87: 1441-1450 and Abeyrathne et al’s 2013 Poultry Science 92: 1091-1097. However, this process was having limitation with colour description and yield.

Therefore, it would be desirable and of paramount important to have a process for the separation of Ovotransferrin, by employing inexpensive, readilyavailable, easy to handle reagents. It would also be desirable to have a process that can be readily scaled up, thereby making it more suitable for industrial scale preparation.

Summary of the Invention

The present invention provides a cost effective, novel and an efficient process for the separation of Ovotransferrin from egg white with higher yields and completely free from EDT/endotoxins.

In one aspect, the present invention provides an improved process for the separation of Ovotransferrin from egg white,which comprises:

i) egg white solution was charged with water, followed by stirring and maintaining the solution pH at acidic medium;

ii) cooled the reaction mixture after filtration, followed by adding salt and citric acid with stirring and centrifugation to obtain solid;

iii) water is added to the above solid, followed by adding sodium chloride, citric acid with stirring;

iv) to the above solid reaction mixture, water is added by stirring;

v) to the above reaction mixture, sodium chloride, citric acid are added, followed by stirring and centrifugation is done to obtain solid;
vi) to the above solid, water is added by stirring and adjusting the pH at basic medium;

vii) EDTA di-metal dihydrate salt is added to the above reaction mixture, followed by series of filtrations, lyophilisation;

viii) ethanol or isopropyl alcohol was added to the lyophilised Ovotransferrin, followed by stirring, filtration and drying to Ovotransferrin free from bacteria and microorganisms.

Detailed Description of the Invention

Accordingly, the present invention provides an improved process for the separation of Ovotransferrin from egg white.

The main aspect of the present invention provides an improved process for the separation of Ovotransferrin from egg white, which is outlined in below process.

step i) egg white solution was charged with water, followed by stirring and maintaining the solution pH at acidic medium;

step ii) cooled the reaction mixture after filtration, followed by adding salt and citric acid with stirring and centrifugation to obtain solid;

step iii) water is added to the above solid, followed by adding sodium chloride, citric acid with stirring;

step iv) to the above solid reaction mixture, water is added by stirring;

step v) to the above reaction mixture, sodium chloride, citric acid are added, followed by stirring and centrifugation is done to obtain solid;
step vi) to the above solid, water is added by stirring and adjusting the pH at basic medium;

step vii) EDTA di-metal dihydrate salt is added to the above reaction mixture, followed by series of filtrations, lyophilisation;

step viii) ethanol or isopropyl alcohol was added to the lyophilised Ovotransferrin, followed by stirring, filtration and drying to Ovotransferrin free from bacteria and microorganisms.

Salt is selected from the group consisting of sodium chloride, ammonium chloride, potassium chloride, magnesium chloride or mixtures.

EXPERIMENTAL PORTION

The details of the invention are given in the examples provided below, which are given to

illustrate the invention only and therefore should not be construed to limit the scope of

*

the invention.

Example-1: Process for the separation of Ovotransferrin from Egg white

97.0 Lt egg white solution was taken in cleaned glass line reactor at 25-30 °C. 97.0 Lt Purified water was charged at ambient temperature followed by stirring to obtained homogeneous solution. Cooled the mixture at 4-10 °C. pH 4.5-5.0 was adjusted by using Hydrochloric acid at temperature 4-10 °C and maintained for 2-3 hours at temperature 4-10 °C Settled the reaction mixture 720 -900 minutesat 4-10 °C. Filtered the reaction mixture through High-flow by using notch filtration at 4-10 °C. Transferred thefiltrate into glass line reactor and cooled the reaction mixture at4-10 °C. Charged 10 Kg ammonium sulphate (Lot-1) followed by 5.0 Kg Citric Acid at 4-10 °C and stirred for 60 minutes at 4-10 °C. Settled the reaction mixture for720-900 minutesat 4-10 °C .Centrifuged the reaction mixture in multiple lots at 3400 RPM and 5.0 kg solid obtained.Transferred the 5 Kg solid into cleaned container at 20-25 °C. 10 lit water (Lot-
2) was charged and stirred for 60 minutesat 5-10 °C Charged 0.75 Kg,ammonium sulphate (Lot-2)into reaction mixture and stirred for 20 minutesat 5-10 °C. Charged 0.375 Kg citric acid at 5-10 °CCentrifiiged the reaction mixture at 3400 RPM for 600-720 minutesat 5-10 °C(solid= 4.350kg).

Transferred the 4.35 Kg solid into cleaned container at 5-10 °C. 8.7 lit water (Lot-2) was charged and stirred for 60 minutes at 5-10 °C Charged 0.26 Kg ammonium sulphate (Lot-2)into reaction mixture and stirred for 20 minutes at 5-10 °C. Charged 0.20 Kg citric acid and stirred at 5-10 °C. Centrifuged the reaction mixture at 3400 RPM for 300-400 minutes at 5-10 °C(solid= 3.99 kg). Charged 3.99 kg the above solid into clean and dry container at 5-10 °C. 8 lit water(lot-4) charged into reaction mixture and stirred the RM for 30 minutes, at 5-10 °C

Charged 60 grams ammonium sulphate (lot-4) into reaction mixture and stirred for 10 minutes at 5-10 °C. Charged 45grams citric acid into Reaction mixture stirred and Centrifuged the RM at 3400 RPM for 300 minutes. Solid 3.6 Kg obtained.Transferred the 3.6 Kg solid into cleaned container at 5-10 °C. 7.2 lit water (Lot-2) was charged and stirred for 30 minutes and centrifuged the RM at 3400 RPM for 240-300 minutes (solid=3.5kg)

Transferred the 3.5 Kg solid into cleaned container at 5-10 °C.17.5 lit chilled water (Lot-2) was charged and stirred for 10 minutes and the pH 10-11 was adjusted by using 20% sodium hydroxide solution (600ml).

* 12 grams EDTA disodium salt dihydrate was charged at 10 °C Filtered the reaction mixture using filter bags with 75 ml water at 10°C .21 Lt reaction mixture was charged into the ultra-filtration tank (PH-10.16, conductivity-5.26 pS and brix value - 4.1). Ultrafiltration Process was started by 10 kDamembrane up to conductivity 0.76 with Bricks value 6.8 (PH-8.7) for 300-600 minutes. Finally unload the 4 L reaction mass from U.F tank (Brix value 7.5) conductivity-0.7 pS Checked the PH-8.65 Filter the solutionthrough cloth followed by paper Solution was loaded into Lyophilization after completing the Lyophilization 390 g Solid unloaded in to transparent LDPE bag.

1.95 Lt Ethanol was charged to the RBF at 25-30 °C. 390 grams Lyophilizedsolid Ovotransferrin was charged and stirred for 240 minutes at 25-30 °C. Filtered and unloaded the solid. Dried the solid at 45-50 OC. 380 g solid was unloaded in Transparent LDPE bag followed by Black LDPE bag. Tested the solid for BET/Endotoxin. Result obtained was in line with FDA guidelines

Example-2: Process for separation of Ovotransferrin from Egg white

97.0 Lt egg white solution was taken in cleaned glass line reactor at 25-30 °C. 97.0 Lt Purified water was charged at ambient temperature followed by stirring to obtained homogeneous solution. Cooled the mixture at 4-10 °C. pH 4.5-5.0 was adjusted by using Hydrochloric acid at temperature 4-10 °C and maintained for 2-3 hrs at temperature 4-10 °C Settled the reaction mixture 720 -900 minutes at 4-10 °C. Filtered the reaction mixture through High-flow by using notch filtration at 4-10 °C. Transferred thefiltrate into glass line reactor and cooled the reaction mixture at4-10 °C. Charged 10 Kg Sodium Chloride (Lot-1) followed by 5.0 Kg Citric Acid at 4-10 °C and stirred for 60 minutes at 4-10 °C. Settled the reaction mixture for 720-900 minutes at 4-10 °C .Centrifuged the reaction mixture in multiple lots at 3400 RPM and 5.0 kg solid obtained.Transferred the 5 Kg solid into cleaned container at 20-25 °C. 10 lit water (Lot-2) was charged and stirred for 60 minutes at 5-10 °C Charged 0.75 Kg,Sodium Chloride (Lot-2)into reaction mixture and stirred for 20 minutes at 5-10 °C. Charged 0.375 Kg citric acid at 5-10 °CCentrifuged the reaction mixture at 3400 RPM for 600-720 minutes at 5-10 °C(solid= 4.350kg).

Transferred the 4.35 Kg solid into cleaned container at 5-10 °C. 8.7 lit water (Lot-2) was charged and stirred for 60 minutes at 5-10 °C Charged 0.26 Kg Sodium Chloride (Lot-2)into reaction mixture and stirred for 20 minutes at 5-10 °C. Charged 0.20 Kg citric acid
and stirred at 5-10 °C. Centrifuged the reaction mixture at 3400 RPM for 300-400 minutes at 5-10 °C(solid= 3.99 kg). Charged 3.99 kg the above solid into clean and dry container at 5-10 °C. 8 lit water(lot-4) charged into reaction mixture and stirred the RM for 30 minutes, at 5-10 °C

Charged 60 g Sodium Chloride (lot-4) into reaction mixture and stirred for 10 minutes, at 5-10 °C. Charged 45g citric acid into Reaction mixture stirred and Centrifuged the RM at 3400 RPM for 300 minutes. Solid 3.6 Kg obtained.Transferred the 3.6 Kg solid into cleaned container at 5-10 °C. 7.2 lit water (Lot-2) was charged and stirred for 30 minutes and centrifuged the RM at 3400 RPM for 240-300 minutes (solid=3.5kg)

Transferred the 3.5 Kg solid into cleaned container at 5-10 °C.17.5 lit chilled water (Lot-2) was charged and stirred for 10 minutes and the pH 10-11 was adjusted by using 20% sodium hydroxide solution (600ml).

12 grams EDTA disodium salt dihydrate was charged at 10 °C Filtered the reaction mixture using filter bags with 75 ml water at 10°C .21 Lt reaction mixture was charged into the ultra-filtration tank (PH-10.16, conductivity-5.26 pS and brix value - 4.1). Ultra-filtration Process was started by 10 kDamembrane up to conductivity 0.76 with Bricks value 6.8 (PH-8.7) for 300-600 minutes. Finally unload the 4 L reaction mass from U.F tank (Brix value 7.5) conductivity-0.7 pS Checked the pH-8.65 Filter the solution through cloth followed by paper Solution was loaded into Lyophilization after completing the Lyophilization 435 g Solid unloaded in to transparent LDPE bag.

2.175 Lt Isopropyl alcohol was charged to the RBF at 25-30 °C. 390 grams Lyophilizedsolid Ovotransferrin was charged and stirred for 240 minutes at 25-30 °C. Filtered and unloaded the solid. Dried the solid at 45-50 OC. 423 g solid was unloaded in Transparent LDPE bag followed by Black LDPE bag. Tested the solid for BET/Endotoxin. Result obtained was in line with FDA guidelines