Title of Invention	"A PROCESS FOR SEPARATION OF SHELL MEMBRANE FROM WET EGG SHELLS"
Abstract	The present invention relates to a process for separation of shell membrane from wet egg shells. The invention in particular relates to the separation of shell membrane from wet shells of hen egg. The treatment of egg shells with EDTA solution may soften the shell and partially detach the membrane from shell; and, hexane may further loosen the adhesion between membrane and shell, simultaneously forming the emulsion containing the released membrane which can be easily separated from egg shell particles. The novelty of the present invention is achieving the separation of membrane from egg shells by milder treatments such as EDTA and hexane which do not affect the protein quality of membrane and obtaining the membrane with minimum contamination from shell particles.

Title of Invention

"A PROCESS FOR SEPARATION OF SHELL MEMBRANE FROM WET EGG SHELLS"

Abstract

The present invention relates to a process for separation of shell membrane from wet egg shells. The invention in particular relates to the separation of shell membrane from wet shells of hen egg. The treatment of egg shells with EDTA solution may soften the shell and partially detach the membrane from shell; and, hexane may further loosen the adhesion between membrane and shell, simultaneously forming the emulsion containing the released membrane which can be easily separated from egg shell particles. The novelty of the present invention is achieving the separation of membrane from egg shells by milder treatments such as EDTA and hexane which do not affect the protein quality of membrane and obtaining the membrane with minimum contamination from shell particles.

Full Text	The present invention relates to a process for separation of shell membrane from wet egg shells. The invention in particular relates to the separation of shell membrane from wet shells of hen egg. India produces over 41 billion eggs per annum and is ranked fourth in the world in egg production. Eight egg processing industries existing in India together process 1.2-1.6 million eggs per day to produce egg powder from liquid egg for export. These industries produce 6-9 MT of egg shell along with membrane per day. Presently the wet egg shells as produced in the industries are used for composting for manure and some quantity is dried and used in animal feeds as a source of calcium. Application of modern technologies would enable to produce high value products from egg shells. Egg shell membrane consists of around 70% protein including 10% collagen. The potential value of collagen from egg shell membrane is huge, especially in biomedical applications, viz., skin grafts, dental implants, angioplasty sleeves, cornea repair, plastic surgery, treatment of osteoporosis and pharmaceuticals as well as food casings and film emulsions. Purified collagen from egg shell membrane costs around US D 1000/g. Since large quantity of egg shells is generated in industries, there is a need for development of a process for separation of membrane from egg shell which can be further used for extraction of collagen. This will also improve the economy of egg processing plants. A few authors have carried out work on separation of membrane from egg shells. Reference may be made to Takahashi et al 1996 (BioSci Biotechnol Biochem 60(8), 1299-1302) wherein soluble egg shell membrane protein (SEP) was prepared from egg shell membrane by the combined treatment of performic acid oxidation (4 or 25°C for 24 h) and pepsin digestion (25°C for 24-72 h) before dialysis against water and lyophilisation. The drawbacks are the use of strong acid, which denatures the valuable protein collagen, and loss of acid-soluble collagen. Reference may be made to Ishikawa et al 1999 (Appl Biochem Biotechnol 77-79. 521-533) wherein egg shell membrane was prepared by mechanical stripping from egg shells which had been immersed in 0.5 M HC1 overnight. The drawbacks are denaturation of collagen and loss of acid-soluble collagen due to treatment with strong acid as mentioned above. Reference may be made to Fernandez et al 2001 (Matrix Biol 19, 793-803) wherein egg shells are treated with 5% tetra sodium ethylenediamine tetra acetate (EDTA) at room temperature for 1 h. This caused partial demineralization, which facilitated partial loosening of membrane from the shell. Membrane was then stripped off with a forced manually. The objective of this study was to understand the mechanism of mineralisation and formation of egg shell. The drawback of the technique is the need for the manual separation of the loosened membrane. Reference may be made to Ishikawa et al 2002 (Bioresource Technol 81(3), 201-206) wherein egg shell membrane was mechanically stripped from the shell by immersing overnight in 0.5 M HC1 followed by immersion in 0.5 M NaOH for 1 h. The drawbacks are denaturation of collagen and loss of acid soluble collagen due to strong acid and alkali. Reference may be made to patent nr US 5053238-1991 by Zeidler et al (Method for shell membrane from hard boiled eggs) wherein boiled eggs are treated with acids and/or alkalies and heated up to 100°C for softening the shell prior to mechanical pealing or to dissolve shell completely. The egg membrane on the boiled egg is removed by treatment with alkali. The objective of the study was to remove the shell of boiled egg without causing damage to the egg white albumen, without affecting the shape, texture and appearance of hard-boiled egg and not the separation of membrane egg shell. The drawback is denaturation and solubilisation of collagen of the membrane due to treatment with acid/alkali and use of high temperature. Reference may be made to patent nr KR 9615880-1996 by Ahn et al (Removing method of egg shell membrane) wherein raw egg shells are stirred with over 3% NaOH or KOH at 60 rpm or more and at 80°C or more followed by neutralizing with 1-3 N HC1 solution, washing and drying to obtain pure egg shells containing 0.1% or less protein. The objective of the work was to obtain pure egg shells and not to recover membrane. The drawbacks are denaturation of collagen and loss of acid soluble collagen of the membrane as mentioned above. Reference may be made to patent nr WO 9841326-1998 by MacNeil JH (Waste egg shell components recovery) wherein the membrane and shell are separated by abrading whereby the linking structure between the shell is at least partially disrupted. Thereafter, the waste shell particles are conveyed to a tank containing liquid, preferably water, and allowed to settle there through. As the waste egg shell particles settle, the particles experience turbulent forces in water, which causes the membrane and shell portions to separate. The drawbacks are partial separation of membrane from shell due to abrading and the need for specialized machinery. Reference may be made to patent nr DE 19841291-1999 by Bartholomes PK (Egg shell separator to remove shell and membrane from boiled eggs) wherein pressurized gas is injected through a needle under the shell of the egg in order to loosen shell from boiled egg. In this method membrane and shell separate from the boiled egg, and membrane still remains attached to the shell. Reference may be made to patent nr US 0166213 Al-2004 by Thoroski JH (Egg shell processing methods and apparatus) wherein egg shells are dried to partially separate the membrane from outer egg shell portions, The membrane is then separated from shell by sieving. The remaining egg shell is impacted to reduce the particle size of at least the outer egg shell material to form smaller particles. The outer egg shell particles are collected to a valve, and the membrane portion is pneumatically removed. The drawback is the loss of membrane that is still attached to the shell particle and contamination of membrane with shell particles. The main object of the present invention is to provide a process for separation of shell membrane from wet egg shells, which obviates the drawbacks as detailed above. Another object of the present invention is to separate membrane from wet egg shell that is minimally contaminated with shell particles. Accordingly, the present invention provides a process for the separation of shell membrane from wet egg shells which comprises: a) breaking eggs in order to obtain wet egg shell fragments; b) soaking wet shell fragments, obtained in step (a), in ethylene diamine tetra acitic acid disodium (Na-EDTA) solution at the ratio of 1:1 (w/v)followed by incubation at a temperature in the range of 24-28 deg. C for a period in the range of 16-24 hours; c) removing ethylene diamine tetra acitic acid disodium (Na-EDTA) solution followed by addition of fresh ethylene diamine tetra acetic acid disodium (Na-EDTA) solution so as to repeat the step (b) twice in order to obtain soaked shell membrane residues; d) adding hexane to soaked shell membrane residue, obtained in step (c), at the ratio in the range of 1:1 to 3:1 (v/w) in order to obtain mixture containing membrane and shell fragments; e) blending the mixture, obtained in the step (d), for a period in the range of 2- 3 minutes followed by standing for a period in the range of 5-10 minutes in order to obtain two distinct layers: organic (hexane) layer at top, containing the membrane in form of emulsion, and aqueous ethylene diamine tetra acetic acid disodium (Na-EDTA) layer at the bottom containing shell fragments; f) separating two layers, obtained in step (e), in order to recover separately organic (hexane) layer, containing membrane emulsion, and aqueous layer containing shell fragments; g) removing aqueous ethylene diamine tetra acetic acid disodium (Na-EDTA) layer from shell fragments followed by drying in order to obtain dried shell grits as by-product; h) separating, by filtration, organic (hexane) layer, containing membrane emulsion, in order to obtain membrane; i) washing membrane in running water for a period in the range of 5-10 minutes in order to obtain water-washed membrane; j) adding acetone to the water-washed membrane, obtained in step (i), at the ratio in the range of 1:1 to 5:1 (v/w acetone :wet shell fragments) followed by holding of the mixture for a period in the range of 5-10 minutes in order to obtain a mixture of acetone and membrane; k) separating, by filtration, acetone from the mixture, obtained in step j), in order to recover acetone-washed membrane; and I) drying the water-washed membrane, obtained in step (k), to obtain desired dried membrane. In an embodiment of the present invention, eggs used in step (a) are eggs laid by members belonging to groups consisting of birds, reptiles, insects and any member of animal kingdom which lays eggs covered with calcareous shell. In an embodiment of the present invention, wet egg shell fragments used in step (a) are by-products of egg processing industry. In an embodiment of the present invention, the yield of dried membrane obtained in step (1) is about 3.2 (w/w) percent wet egg shell fragments. In an embodiment of the present invention, wherein the yield of dried shell grits obtained as by-product in step (g) is at about 55.3 (w/w) percent wet egg shell fragments. In an embodiment of the present invention, wherein dried membrane obtained in step (1) has protein and collagen having purity in the range of 87.3-99.7 percent. In an embodiment of the present invention, the wet egg shells obtained by breaking the eggs are soaked in 2.0 - 8.0% Na-EDTA solution (1 : 2.5 to 7.5) for 16-24 h at ambient temperature (26 ± 2°C) and the process repeated twice. In another embodiment of the present invention, hexane (1 : 1 to 3.0) is added to the mixture of egg shells and Na-EDTA solution, and blend for 2-3 min, and allowed to stand for 5-10 min to separate organic and aqueous layer. In yet another embodiment of the present invention, hexane layer containing separated membrane in the form of emulsion is decanted. In still another embodiment of the present invention, the hexane layer is filtered to separate spent hexane and recover membrane as residue. In still another embodiment of the present invention, the recovered membrane is washed in running water for 5-10 min followed by soaking in acetone (1 : 1 to 5) for 5-10 min and filtered to get the membrane. In still another embodiment of the present invention, the separated membrane is dried at 40-55°C for 20 - 60 min to obtain dried membrane. The treatment of wet egg shells to recover membrane is presented in the flow chart. Wet egg shells obtained by breaking eggs were soaked 5% Ethylene Diamine Tetra Acetic acid disodium (Na-EDTA) in the ratio of shell to solution of 1 : 5 and held at ambient temperature (26 ± 2°C) for 24 h. The EDTA solution was decanted and the shells were soaked again in fresh solution of 5% Na-EDTA in the ratio of shell to solution of 1 : 5. After soaking for 24 h at 26 + 2°C, the solution was decanted and replaced with fresh EDTA solution. After holding at 26 ± 2°C for 24 h, hexane (half the quantity of EDTA solution) was added and blend for 3 min. The mixture was allowed to stand for 5 min to separate organic and aqueous layers. The hexane layer containing separated membrane in the form of emulsion was decanted and filtered to separate membrane as residue. The residue was then washed in running water for 5 min followed by soaking in acetone (equal the quantity of hexane), filtered and dried in cross flow drier at 50°C for 30 min, to obtain dried membrane. (Figure removed) The treatment of egg shells with EDTA solution may soften the shell and partially detach the membrane from shell; and, hexane may further loosen the adhesion between membrane and shell, simultaneously forming the emulsion containing the released membrane which can be easily separated from egg shell particles. The novelty of the present invention is achieving the separation of membrane from egg shells by milder treatments such as EDTA and hexane which do not affect the protein quality of membrane and obtaining the membrane with minimum contamination from shell particles. The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention Example 1 Wet egg shells (50.5 g) obtained by breaking eggs were soaked in 250 ml of 5% Na-EDTA solution (1:5) and held at ambient temperature (26 ± 2°C) for 24 h. The EDTA solution was decanted and the shells were soaked again in 250 ml of fresh solution of 5% Na-EDTA (1 : 5). After soaking for 24 h at 26 + 2°C, the solution was decanted and replaced with 250 ml of fresh EDTA solution. After holding at 26±2°C for 24 h, 125 ml of hexane (half the quantity of EDTA solution) was added and blend for 3 min. The mixture was allowed to stand for 5 min to separate organic and aqueous layers. The hexane layer containing separated membrane in the form of emulsion was decanted and filtered to separate membrane as residue. The residue was then washed in running water for 5 min followed by soaking in 125 ml acetone, filtered and dried in cross flow drier at 50°C for 30 min, to obtain 1.62 g of dried membrane. The shell residue in EDTA solution was separated by filtration. The residue was then dried in cross flow air drier at 50°C for 15 h to obtain 27.9 g dried shell grits. The yields of dried membrane and dried shell grits were calculated as % of wet egg shells. The protein and collagen contents were estimated by standard methods (AOAC 1995) in the dried membrane, dried shell residue as well as in the manually separated and dried membrane from wet egg shells. The purity of the recovered membrane was assessed by comparing protein and collagen with that of manually separated membrane from wet egg shells. (Table removed) The results presented in table 1 indicate that the yields of dried membrane and dried shell residue were 3.2% and 55.3% respectively. Protein and collagen recovery respectively were 69.8% and 79.1% for dried membrane and 26.3% and 1.8% for dried shell residue. The protein content of recovered and dried membrane was 74.2% as against 78.9% for manually separated and dried membrane. Similarly, the collagen content of recovered membrane was 2.0949% as against 2.3991% for manually separated and dried membrane. Example 2 Wet egg shells (106.4 g) were soaked in 500 ml of 5% Na-EDTA solution (1 : 5) and held at 26 ± 2°C for 24 h. Repeated soaking of shells in fresh EDTA solution was carried out as explained in example 1. To the mixture of shells and EDTA solution, 250 ml of hexane was added and blend for 4 min. The mixture was allowed to stand for 10 min to separate organic and aqueous layers. The hexane layer was decanted, filtered, residue washed with water, soaked in acetone and dried as mentioned in example 1 to obtain 3.1 g of membrane. The egg shell residue in EDTA solution was separated by filtration, the residue dried in cross flow air drier at 50°C for 2 h to obtain 49.1 g dried shell grits. The yields of dried membrane and shell grits, recovery of protein and collagen in dried membrane and grits was determined as detailed in example 1. (Table removed) The results of table 2 revealed that yields of dried membrane and grits were 2.9% and 46.1% respectively. The recovery of protein and collagen respectively was 62.1% and 92.9% for dried membrane and 36.6% and 3.3% for dried shell grits. The protein and collagen contents of solvent-treated and dried membrane were 85.5% and 2.42% as against 86.1% and 2.53% respectively for manually separated and dried membrane. These observations are similar to those made in example 1. Example 3 Wet egg shells (55.8g) obtained as mentioned in example 1 were soaked in 280 ml of 5% Na-EDTA solution (1:5) for 24 h at 26 + 2°C. Treatment with EDTA followed by hexane was repeated as in example 1. Dried membrane (1.51 g) and dried egg shell grits (25.7 g) were obtained following the procedure detailed earlier. The samples were analyzed for yield, protein and collagen contents. (Table removed) The yields of dried membrane and shell grits were 2.7% and 46.1 % respectively (Table 3). The recovery of protein and collagen contents respectively was 59.8% and 94.2% for dried membrane and 40.6% and 3.1% for dried grits. The protein and collagen contents for solvent treated and dried membrane were 88.4% and 2.6% as against 89.5% and 2.6% respectively for manually separated and dried membranes as also observed in earlier examples. Example 4 Wet egg shells (118.5 g) were soaked in 590 ml of 5% Na-EDTA solution (1.5), treatment repeated again, membrane in hexane layer and shell grits in aqueous layer were separated and dried as mentioned in example 1. Dried membrane (3.56 g), dried shell grits (64.6 g) and manually separated and dried membrane were analysed for collagen and protein content as detailed in example 1. (Table removed) The yield of dried membrane and shell grits were 3.0% and 54.5% respectively (Table 4). The recovery of protein and collagen contents respectively was 67.3% and 88.4% for membrane and 25.4% and 2.5% for grits. The protein contents for solvent-treated and dried membrane were 84% and 2.44% as compared to 85.5% and 2.61% for manually separated and dried membrane. Similar observations were made in Examples 1, 2 and 3. The main advantages of the present invention are: 1. Loosening the membrane from egg shell by milder treatments with EDTA and hexane. 2. Greater purity of separated membrane as measured by protein and collagen contents (87.3 to 99.7% of manually separated membrane). 3. Less contamination of separated membrane with shell particles as indicated by the closeness of protein and collagen content to that of manually separated membrane. remaining egg shell is impacted to reduce the particle size of at least the outer egg shell material to form smaller particles. The outer egg shell particles are collected to a valve, and the membrane portion is pneumatically removed. The drawback is the loss of membrane that is still attached to the shell particle and contamination of membrane with shell particles. The main object of the present invention is to provide a process for separation of shell membrane from wet egg shells, which obviates the drawbacks as detailed above. Another object of the present invention is to separate membrane from wet egg shell that is minimally contaminated with shell particles. Accordingly, the present invention provides a process for the separation of shell membrane from wet egg shells which comprises: a) breaking eggs in order to obtain wet egg shell fragments; b) soaking wet shell fragments, obtained in step (a), in ethylene diamine tetra acitic acid disodium (Na-EDTA) solution at the ratio of 1:1 (w/v)followed by incubation at a temperature in the range of 24-28 deg. C for a period in the range of 16-24 hours; c) removing ethylene diamine tetra acitic acid disodium (Na-EDTA) solution followed by addition of fresh ethylene diamine tetra acetic acid disodium (Na-EDTA) solution so as to repeat the step (b) twice in order to obtain soaked shell membrane residues; d) adding hexane to soaked shell membrane residue, obtained in step (c), at the ratio in the range of 1:1 to 3:1 (v/w) in order to obtain mixture containing membrane and shell fragments; e) blending the mixture, obtained in the step (d), for a period in the range of 2- 3 minutes followed by standing for a period in the range of 5-10 minutes in order to obtain two distinct layers: organic (hexane) layer at top, containing the membrane in form of emulsion, and aqueous ethylene diamine tetra acetic acid disodium (Na-EDTA) layer at the bottom containing shell fragments; f) separating two layers, obtained in step (e), in order to recover separately organic (hexane) layer, containing membrane emulsion, and aqueous layer containing shell fragments; g) removing aqueous ethylene diamine tetra acetic acid disodium (Na-EDTA) layer from shell fragments followed by drying in order to obtain dried shell grits as by-product; h) separating, by filtration, organic (hexane) layer, containing membrane emulsion, in order to obtain membrane; i) washing membrane in running water for a period in the range of 5-10 minutes in order to obtain water-washed membrane; j) adding acetone to the water-washed membrane, obtained in step (i), at the ratio in the range of 1:1 to 5:1 (v/w acetone :wet shell fragments) followed by holding of the mixture for a period in the range of 5-10 minutes in order to obtain a mixture of acetone and membrane; k) separating, by filtration, acetone from the mixture, obtained in step j), in order to recover acetone-washed membrane; and I) drying the water-washed membrane, obtained in step (k), to obtain desired dried membrane. In an embodiment of the present invention, eggs used in step (a) are eggs laid by members belonging to groups consisting of birds, reptiles, insects and any member of animal kingdom which lays eggs covered with calcareous shell.

Full Text

The present invention relates to a process for separation of shell membrane from wet egg shells. The invention in particular relates to the separation of shell membrane from wet shells of hen egg.
India produces over 41 billion eggs per annum and is ranked fourth in the world in egg production. Eight egg processing industries existing in India together process 1.2-1.6 million eggs per day to produce egg powder from liquid egg for export. These industries produce 6-9 MT of egg shell along with membrane per day. Presently the wet egg shells as produced in the industries are used for composting for manure and some quantity is dried and used in animal feeds as a source of calcium. Application of modern technologies would enable to produce high value products from egg shells. Egg shell membrane consists of around 70% protein including 10% collagen. The potential value of collagen from egg shell membrane is huge, especially in biomedical applications, viz., skin grafts, dental implants, angioplasty sleeves, cornea repair, plastic surgery, treatment of osteoporosis and pharmaceuticals as well as food casings and film emulsions. Purified collagen from egg shell membrane costs around US D 1000/g. Since large quantity of egg shells is generated in industries, there is a need for development of a process for separation of membrane from egg shell which can be further used for extraction of collagen. This will also improve the economy of egg processing plants. A few authors have carried out work on separation of membrane from egg shells.
Reference may be made to Takahashi et al 1996 (BioSci Biotechnol Biochem 60(8), 1299-1302) wherein soluble egg shell membrane protein (SEP) was prepared from egg shell membrane by the combined treatment of performic acid oxidation (4 or 25°C for 24 h) and pepsin digestion (25°C for 24-72 h) before dialysis against water and lyophilisation. The drawbacks are the use of strong acid, which denatures the valuable protein collagen, and loss of acid-soluble collagen.
Reference may be made to Ishikawa et al 1999 (Appl Biochem Biotechnol 77-79. 521-533) wherein egg shell membrane was prepared by mechanical stripping from egg shells which had been immersed in 0.5 M HC1 overnight. The drawbacks are denaturation of collagen and loss of acid-soluble collagen due to treatment with strong acid as mentioned above.
Reference may be made to Fernandez et al 2001 (Matrix Biol 19, 793-803) wherein egg shells are treated with 5% tetra sodium ethylenediamine tetra acetate (EDTA) at room temperature for 1 h. This caused partial demineralization, which facilitated partial loosening of membrane from the shell. Membrane was then stripped off with a forced manually. The objective of this study was to understand the mechanism of mineralisation and formation of egg shell. The drawback of the technique is the need for the manual separation of the loosened membrane.
Reference may be made to Ishikawa et al 2002 (Bioresource Technol 81(3), 201-206) wherein egg shell membrane was mechanically stripped from the
shell by immersing overnight in 0.5 M HC1 followed by immersion in 0.5 M NaOH for 1 h. The drawbacks are denaturation of collagen and loss of acid soluble collagen due to strong acid and alkali.
Reference may be made to patent nr US 5053238-1991 by Zeidler et al (Method for shell membrane from hard boiled eggs) wherein boiled eggs are treated with acids and/or alkalies and heated up to 100°C for softening the shell prior to mechanical pealing or to dissolve shell completely. The egg membrane on the boiled egg is removed by treatment with alkali. The objective of the study was to remove the shell of boiled egg without causing damage to the egg white albumen, without affecting the shape, texture and appearance of hard-boiled egg and not the separation of membrane egg shell. The drawback is denaturation and solubilisation of collagen of the membrane due to treatment with acid/alkali and use of high temperature.
Reference may be made to patent nr KR 9615880-1996 by Ahn et al (Removing method of egg shell membrane) wherein raw egg shells are stirred with over 3% NaOH or KOH at 60 rpm or more and at 80°C or more followed by neutralizing with 1-3 N HC1 solution, washing and drying to obtain pure egg shells containing 0.1% or less protein. The objective of the work was to obtain pure egg shells and not to recover membrane. The drawbacks are denaturation of collagen and loss of acid soluble collagen of the membrane as mentioned above.
Reference may be made to patent nr WO 9841326-1998 by MacNeil JH (Waste egg shell components recovery) wherein the membrane and shell are
separated by abrading whereby the linking structure between the shell is at least partially disrupted. Thereafter, the waste shell particles are conveyed to a tank containing liquid, preferably water, and allowed to settle there through. As the waste egg shell particles settle, the particles experience turbulent forces in water, which causes the membrane and shell portions to separate. The drawbacks are partial separation of membrane from shell due to abrading and the need for specialized machinery.
Reference may be made to patent nr DE 19841291-1999 by Bartholomes PK (Egg shell separator to remove shell and membrane from boiled eggs) wherein pressurized gas is injected through a needle under the shell of the egg in order to loosen shell from boiled egg. In this method membrane and shell separate from the boiled egg, and membrane still remains attached to the shell.
Reference may be made to patent nr US 0166213 Al-2004 by Thoroski JH (Egg shell processing methods and apparatus) wherein egg shells are dried to partially separate the membrane from outer egg shell portions, The membrane is then separated from shell by sieving. The
remaining egg shell is impacted to reduce the particle size of at least the outer egg shell material to form smaller particles. The outer egg shell particles are collected to a valve, and the membrane portion is pneumatically removed. The drawback is the loss of membrane that is still attached to the shell particle and contamination of membrane with shell particles.
The main object of the present invention is to provide a process for separation of shell membrane from wet egg shells, which obviates the drawbacks as detailed above.
Another object of the present invention is to separate membrane from wet egg shell that is minimally contaminated with shell particles.
Accordingly, the present invention provides a process for the separation of shell membrane from wet egg shells which comprises:
a) breaking eggs in order to obtain wet egg shell fragments;
b) soaking wet shell fragments, obtained in step (a), in ethylene diamine tetra acitic acid disodium (Na-EDTA) solution at the ratio of 1:1 (w/v)followed by incubation at a temperature in the range of 24-28 deg. C for a period in the range of 16-24 hours;

c) removing ethylene diamine tetra acitic acid disodium (Na-EDTA) solution followed by addition of fresh ethylene diamine tetra acetic acid disodium (Na-EDTA) solution so as to repeat the step (b) twice in order to obtain soaked shell membrane residues;
d) adding hexane to soaked shell membrane residue, obtained in step (c), at the ratio in the range of 1:1 to 3:1 (v/w) in order to obtain mixture containing membrane and shell fragments;
e) blending the mixture, obtained in the step (d), for a period in the range of 2- 3 minutes followed by standing for a period in the range of 5-10 minutes in order to obtain two distinct layers: organic (hexane) layer at top, containing the membrane in form of emulsion, and aqueous ethylene diamine tetra acetic acid disodium (Na-EDTA) layer at the bottom containing shell fragments;
f) separating two layers, obtained in step (e), in order to recover separately organic (hexane) layer, containing membrane emulsion, and aqueous layer containing shell fragments;
g) removing aqueous ethylene diamine tetra acetic acid disodium (Na-EDTA) layer from shell fragments followed by drying in order to obtain dried shell grits as by-product;
h) separating, by filtration, organic (hexane) layer, containing
membrane emulsion, in order to obtain membrane;
i) washing membrane in running water for a period in the range of
5-10 minutes in order to obtain water-washed membrane;
j) adding acetone to the water-washed membrane, obtained in step
(i), at the ratio in the range of 1:1 to 5:1 (v/w acetone :wet shell
fragments) followed by holding of the mixture for a period in the
range of 5-10 minutes in order to obtain a mixture of acetone and
membrane;
k) separating, by filtration, acetone from the mixture, obtained in
step j), in order to recover acetone-washed membrane; and
I) drying the water-washed membrane, obtained in step (k), to obtain
desired dried membrane.
In an embodiment of the present invention, eggs used in step
(a) are eggs laid by members belonging to groups consisting of birds,
reptiles, insects and any member of animal kingdom which lays eggs
covered with calcareous shell.
In an embodiment of the present invention, wet egg shell fragments used in step (a) are by-products of egg processing industry.
In an embodiment of the present invention, the yield of dried membrane obtained in step (1) is about 3.2 (w/w) percent wet egg shell fragments.
In an embodiment of the present invention, wherein the yield of dried shell grits
obtained as by-product in step (g) is at about 55.3 (w/w) percent wet egg shell fragments.
In an embodiment of the present invention, wherein dried membrane obtained in step (1) has protein and collagen having purity in the range of 87.3-99.7 percent.
In an embodiment of the present invention, the wet egg shells obtained by breaking the eggs are soaked in 2.0 - 8.0% Na-EDTA solution (1 : 2.5 to 7.5) for 16-24 h at ambient temperature (26 ± 2°C) and the process repeated twice.
In another embodiment of the present invention, hexane (1 : 1 to 3.0) is added to the mixture of egg shells and Na-EDTA solution, and blend for 2-3 min, and allowed to stand for 5-10 min to separate organic and aqueous layer.
In yet another embodiment of the present invention, hexane layer containing separated membrane in the form of emulsion is decanted.
In still another embodiment of the present invention, the hexane layer is filtered to separate spent hexane and recover membrane as residue.
In still another embodiment of the present invention, the recovered membrane is washed in running water for 5-10 min followed by soaking in acetone (1 : 1 to 5) for 5-10 min and filtered to get the membrane.

In still another embodiment of the present invention, the separated membrane is dried at 40-55°C for 20 - 60 min to obtain dried membrane.
The treatment of wet egg shells to recover membrane is presented in the flow chart. Wet egg shells obtained by breaking eggs were soaked 5% Ethylene Diamine Tetra Acetic acid disodium (Na-EDTA) in the ratio of shell to solution of 1 : 5 and held at ambient temperature (26 ± 2°C) for 24 h. The EDTA solution was decanted and the shells were soaked again in fresh solution of 5% Na-EDTA in the ratio of shell to solution of 1 : 5. After soaking for 24 h at 26 + 2°C, the solution was decanted and replaced with fresh EDTA solution. After holding at 26 ± 2°C for 24 h, hexane (half the quantity of EDTA solution) was added and blend for 3 min. The mixture was allowed to stand for 5 min to separate organic and aqueous layers. The hexane layer containing separated membrane in the form of emulsion was decanted and filtered to separate membrane as residue. The residue was then washed in running water for 5 min followed by soaking in acetone (equal the quantity of hexane), filtered and dried in cross flow drier at 50°C for 30 min, to obtain dried membrane.
(Figure removed)
The treatment of egg shells with EDTA solution may soften the shell and partially detach the membrane from shell; and, hexane may further loosen the adhesion between membrane and shell, simultaneously forming the emulsion containing the released membrane which can be easily separated from egg shell particles.
The novelty of the present invention is achieving the separation of membrane from egg shells by milder treatments such as EDTA and hexane which do not affect the protein quality of membrane and obtaining the membrane with minimum contamination from shell particles.
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention
Example 1
Wet egg shells (50.5 g) obtained by breaking eggs were soaked in 250 ml of 5% Na-EDTA solution (1:5) and held at ambient temperature (26 ± 2°C) for 24 h. The EDTA solution was decanted and the shells were soaked again in 250 ml of fresh solution of 5% Na-EDTA (1 : 5). After soaking for 24 h at 26 + 2°C, the solution was decanted and replaced with 250 ml of fresh EDTA solution. After holding at 26±2°C for 24 h, 125 ml of hexane (half the quantity of EDTA solution) was added and blend for 3 min. The mixture was allowed to stand for 5 min to separate organic and aqueous layers. The hexane layer containing separated membrane in the form of emulsion was decanted and filtered to separate membrane as residue. The residue was then washed in running water for 5 min
followed by soaking in 125 ml acetone, filtered and dried in cross flow drier at 50°C for 30 min, to obtain 1.62 g of dried membrane. The shell residue in EDTA solution was separated by filtration. The residue was then dried in cross flow air drier at 50°C for 15 h to obtain 27.9 g dried shell grits.
The yields of dried membrane and dried shell grits were calculated as % of wet egg shells. The protein and collagen contents were estimated by standard methods (AOAC 1995) in the dried membrane, dried shell residue as well as in the manually separated and dried membrane from wet egg shells. The purity of the recovered membrane was assessed by comparing protein and collagen with that of manually separated membrane from wet egg shells.
(Table removed)
The results presented in table 1 indicate that the yields of dried membrane and dried shell residue were 3.2% and 55.3% respectively. Protein and collagen recovery respectively were 69.8% and 79.1% for dried membrane and 26.3% and 1.8% for dried shell residue. The protein content of recovered and dried
membrane was 74.2% as against 78.9% for manually separated and dried membrane. Similarly, the collagen content of recovered membrane was 2.0949% as against 2.3991% for manually separated and dried membrane.
Example 2
Wet egg shells (106.4 g) were soaked in 500 ml of 5% Na-EDTA solution (1 : 5) and held at 26 ± 2°C for 24 h. Repeated soaking of shells in fresh EDTA solution was carried out as explained in example 1. To the mixture of shells and EDTA solution, 250 ml of hexane was added and blend for 4 min. The mixture was allowed to stand for 10 min to separate organic and aqueous layers. The hexane layer was decanted, filtered, residue washed with water, soaked in acetone and dried as mentioned in example 1 to obtain 3.1 g of membrane. The egg shell residue in EDTA solution was separated by filtration, the residue dried in cross flow air drier at 50°C for 2 h to obtain 49.1 g dried shell grits. The yields of dried membrane and shell grits, recovery of protein and collagen in dried membrane and grits was determined as detailed in example 1.
(Table removed)
The results of table 2 revealed that yields of dried membrane and grits were 2.9% and 46.1% respectively. The recovery of protein and collagen respectively was 62.1% and 92.9% for dried membrane and 36.6% and 3.3% for dried shell grits. The protein and collagen contents of solvent-treated and dried membrane were 85.5% and 2.42% as against 86.1% and 2.53% respectively for manually separated and dried membrane. These observations are similar to those made in example 1.
Example 3
Wet egg shells (55.8g) obtained as mentioned in example 1 were soaked in 280 ml of 5% Na-EDTA solution (1:5) for 24 h at 26 + 2°C. Treatment with EDTA followed by hexane was repeated as in example 1. Dried membrane (1.51 g) and dried egg shell grits (25.7 g) were obtained following the procedure detailed earlier. The samples were analyzed for yield, protein and collagen contents.
(Table removed)
The yields of dried membrane and shell grits were 2.7% and 46.1 % respectively (Table 3). The recovery of protein and collagen contents respectively was 59.8% and 94.2% for dried membrane and 40.6% and 3.1% for dried grits. The protein and collagen contents for solvent treated and dried membrane were 88.4% and 2.6% as against 89.5% and 2.6% respectively for manually separated and dried membranes as also observed in earlier examples.
Example 4
Wet egg shells (118.5 g) were soaked in 590 ml of 5% Na-EDTA solution (1.5), treatment repeated again, membrane in hexane layer and shell grits in aqueous layer were separated and dried as mentioned in example 1. Dried membrane (3.56 g), dried shell grits (64.6 g) and manually separated and dried membrane were analysed for collagen and protein content as detailed in example 1.
(Table removed)
The yield of dried membrane and shell grits were 3.0% and 54.5% respectively (Table 4). The recovery of protein and collagen contents respectively was 67.3% and 88.4% for membrane and 25.4% and 2.5% for grits. The protein contents for solvent-treated and dried membrane were 84% and 2.44% as compared to 85.5% and 2.61% for manually separated and dried membrane. Similar observations were made in Examples 1, 2 and 3.
The main advantages of the present invention are:
1. Loosening the membrane from egg shell by milder treatments with EDTA
and hexane.
2. Greater purity of separated membrane as measured by protein and collagen
contents (87.3 to 99.7% of manually separated membrane).
3. Less contamination of separated membrane with shell particles as
indicated by the closeness of protein and collagen content to that of
manually separated membrane.

remaining egg shell is impacted to reduce the particle size of at least the outer egg shell material to form smaller particles. The outer egg shell particles are collected to a valve, and the membrane portion is pneumatically removed. The drawback is the loss of membrane that is still attached to the shell particle and contamination of membrane with shell particles.
The main object of the present invention is to provide a process for separation of shell membrane from wet egg shells, which obviates the drawbacks as detailed above.
Another object of the present invention is to separate membrane from wet egg shell that is minimally contaminated with shell particles.
Accordingly, the present invention provides a process for the separation of shell membrane from wet egg shells which comprises:
a) breaking eggs in order to obtain wet egg shell fragments;
b) soaking wet shell fragments, obtained in step (a), in ethylene diamine tetra acitic acid disodium (Na-EDTA) solution at the ratio of 1:1 (w/v)followed by incubation at a temperature in the range of 24-28 deg. C for a period in the range of 16-24 hours;

c) removing ethylene diamine tetra acitic acid disodium (Na-EDTA) solution followed by addition of fresh ethylene diamine tetra acetic acid disodium (Na-EDTA) solution so as to repeat the step (b) twice in order to obtain soaked shell membrane residues;
d) adding hexane to soaked shell membrane residue, obtained in step (c), at the ratio in the range of 1:1 to 3:1 (v/w) in order to obtain mixture containing membrane and shell fragments;
e) blending the mixture, obtained in the step (d), for a period in the range of 2- 3 minutes followed by standing for a period in the range of 5-10 minutes in order to obtain two distinct layers: organic (hexane) layer at top, containing the membrane in form of emulsion, and aqueous ethylene diamine tetra acetic acid disodium (Na-EDTA) layer at the bottom containing shell fragments;
f) separating two layers, obtained in step (e), in order to recover separately organic (hexane) layer, containing membrane emulsion, and aqueous layer containing shell fragments;
g) removing aqueous ethylene diamine tetra acetic acid disodium (Na-EDTA) layer from shell fragments followed by drying in order to obtain dried shell grits as by-product;
h) separating, by filtration, organic (hexane) layer, containing
membrane emulsion, in order to obtain membrane;
i) washing membrane in running water for a period in the range of
5-10 minutes in order to obtain water-washed membrane;
j) adding acetone to the water-washed membrane, obtained in step
(i), at the ratio in the range of 1:1 to 5:1 (v/w acetone :wet shell
fragments) followed by holding of the mixture for a period in the
range of 5-10 minutes in order to obtain a mixture of acetone and
membrane;
k) separating, by filtration, acetone from the mixture, obtained in
step j), in order to recover acetone-washed membrane; and
I) drying the water-washed membrane, obtained in step (k), to obtain
desired dried membrane.
In an embodiment of the present invention, eggs used in step
(a) are eggs laid by members belonging to groups consisting of birds,
reptiles, insects and any member of animal kingdom which lays eggs
covered with calcareous shell.

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Patent Number

258838

Indian Patent Application Number

315/DEL/2006

PG Journal Number

07/2014

Publication Date

14-Feb-2014

Grant Date

10-Feb-2014

Date of Filing

03-Feb-2006

Name of Patentee

COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH.

Applicant Address

ANUSANDHAN BHAWAN, RAFI MARG, NEW DELHI-110 001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	NAKKARIKE MANJABHAT SACHINDRA	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE MYSORE, 570 020, INDIA.
2	NARAYAN BHASKAR	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE MYSORE, 570 020, INDIA.
3	PATIRAM ZITUJI SAKHARE	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE MYSORE, 570 020, INDIA.AC
4	NAMADEV SUBBANNA MAHENDRAKAR	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE MYSORE, 570 020, INDIA.AC

PCT International Classification Number

B13B 5/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA