CA2712375A1 - A method for commercial isolation of egg yolk igy, its product, and uses of the product - Google Patents
A method for commercial isolation of egg yolk igy, its product, and uses of the product Download PDFInfo
- Publication number
- CA2712375A1 CA2712375A1 CA2712375A CA2712375A CA2712375A1 CA 2712375 A1 CA2712375 A1 CA 2712375A1 CA 2712375 A CA2712375 A CA 2712375A CA 2712375 A CA2712375 A CA 2712375A CA 2712375 A1 CA2712375 A1 CA 2712375A1
- Authority
- CA
- Canada
- Prior art keywords
- igy
- fraction
- isolate
- water soluble
- soluble fraction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 102000002322 Egg Proteins Human genes 0.000 title claims abstract description 60
- 108010000912 Egg Proteins Proteins 0.000 title claims abstract description 60
- 235000013345 egg yolk Nutrition 0.000 title claims abstract description 56
- 210000002969 egg yolk Anatomy 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims description 72
- 238000002955 isolation Methods 0.000 title description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 56
- 239000000203 mixture Substances 0.000 claims abstract description 45
- 238000001179 sorption measurement Methods 0.000 claims abstract description 28
- 238000005342 ion exchange Methods 0.000 claims abstract description 25
- 238000005374 membrane filtration Methods 0.000 claims abstract description 22
- 239000012141 concentrate Substances 0.000 claims abstract description 20
- 238000003756 stirring Methods 0.000 claims abstract description 16
- 235000013601 eggs Nutrition 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 241000271566 Aves Species 0.000 claims abstract description 12
- 239000003463 adsorbent Substances 0.000 claims abstract description 10
- 238000000605 extraction Methods 0.000 claims abstract description 9
- 238000001914 filtration Methods 0.000 claims abstract description 9
- 239000011324 bead Substances 0.000 claims abstract description 8
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000007865 diluting Methods 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 7
- 210000000969 egg white Anatomy 0.000 claims abstract description 7
- 235000014103 egg white Nutrition 0.000 claims abstract description 7
- 238000000746 purification Methods 0.000 claims abstract description 7
- 239000007787 solid Substances 0.000 claims description 31
- 239000000243 solution Substances 0.000 claims description 25
- 238000010790 dilution Methods 0.000 claims description 21
- 239000012895 dilution Substances 0.000 claims description 21
- 238000005119 centrifugation Methods 0.000 claims description 18
- 239000000047 product Substances 0.000 claims description 13
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 claims description 10
- 229910001415 sodium ion Inorganic materials 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 8
- 239000002244 precipitate Substances 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 3
- 108010001160 IgY Proteins 0.000 description 70
- 108060003951 Immunoglobulin Proteins 0.000 description 10
- 102000018358 immunoglobulin Human genes 0.000 description 10
- 229940072221 immunoglobulins Drugs 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 241000287828 Gallus gallus Species 0.000 description 5
- 210000000991 chicken egg Anatomy 0.000 description 5
- 235000013330 chicken meat Nutrition 0.000 description 5
- 238000005377 adsorption chromatography Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 4
- 238000005191 phase separation Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 208000002874 Acne Vulgaris Diseases 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 239000004254 Ammonium phosphate Substances 0.000 description 2
- 241000272517 Anseriformes Species 0.000 description 2
- 239000005635 Caprylic acid (CAS 124-07-2) Substances 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 206010000496 acne Diseases 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 2
- 235000019289 ammonium phosphates Nutrition 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 239000001166 ammonium sulphate Substances 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 239000013043 chemical agent Substances 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 2
- 235000015872 dietary supplement Nutrition 0.000 description 2
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 2
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229960002446 octanoic acid Drugs 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 235000013594 poultry meat Nutrition 0.000 description 2
- 238000001223 reverse osmosis Methods 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010017964 Gastrointestinal infection Diseases 0.000 description 1
- 241000617996 Human rotavirus Species 0.000 description 1
- 241000286209 Phasianidae Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000000688 enterotoxigenic effect Effects 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 230000002535 lyotropic effect Effects 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 238000001728 nano-filtration Methods 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000009928 pasteurization Methods 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- -1 polyethylene Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/08—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from eggs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Polymers & Plastics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Animal Husbandry (AREA)
- Immunology (AREA)
- Meat, Egg Or Seafood Products (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
A method for extraction and purification of IgY from immune avian egg yolk comprising diluting a mixture of liquid egg yolk and liquid egg white in treated water;
stirring the diluted mixture to obtain an egg mixture; separating the egg mixture into a first water soluble fraction, an unseparated mixture fraction, and a first separated lipophilic fraction;
separating the first water soluble fraction; the unseparated mixture fraction, and the first separated lipophilic fraction; centrifuging the unseparated mixture fraction until it separates into a second water soluble fraction and a second separated lipophilic fraction;
adding the second water soluble fraction to the first water soluble fraction to form a combined water soluble fraction; passing the combined water soluble fraction through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY;
eluting the adsorbed IgY of from the ion exchange adsorption column with an eluting solution to obtain an eluant; filtering the eluant of through a first membrane filtration system to obtain a 4% to 25% IgY concentrate; and drying the IgY concentrate to obtain a 40% to 98% IgY isolate, as well as a food-safe IgY isolate obtained and use of a food-safe IgY
isolate in an edible product.
stirring the diluted mixture to obtain an egg mixture; separating the egg mixture into a first water soluble fraction, an unseparated mixture fraction, and a first separated lipophilic fraction;
separating the first water soluble fraction; the unseparated mixture fraction, and the first separated lipophilic fraction; centrifuging the unseparated mixture fraction until it separates into a second water soluble fraction and a second separated lipophilic fraction;
adding the second water soluble fraction to the first water soluble fraction to form a combined water soluble fraction; passing the combined water soluble fraction through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY;
eluting the adsorbed IgY of from the ion exchange adsorption column with an eluting solution to obtain an eluant; filtering the eluant of through a first membrane filtration system to obtain a 4% to 25% IgY concentrate; and drying the IgY concentrate to obtain a 40% to 98% IgY isolate, as well as a food-safe IgY isolate obtained and use of a food-safe IgY
isolate in an edible product.
Description
A METHOD FOR COMMERCIAL ISOLATION OF EGG YOLK IgY, ITS PRODUCT, AND USES OF THE PRODUCT
FIELD OF THE INVENTION
The present invention relates to a method for isolation of egg yolk immunoglobulins. In particular, the present invention relates to a method for isolation of highly purified immunoglobulins from avian egg yolk on a commercial scale.
BACKGROUND OF THE INVENTION
Egg yolk separated from eggs obtained from poultry is an excellent source of polyclonal antibodies. For example, immunoglobulin Y ('IgY') may be isolated from chicken egg yolk.
There are several advantages to obtaining immunoglobulins from chicken egg yolks. Use of egg yolk does not require blood extraction, as is the case with mammalian antibody sources, so it may be easier to comply with animal protection regulations.
Chicken eggs also provide a continual supply of source material. The extent of non-specific binding by IgY is reduced, relative to immunoglobulin sourced from mammals, as chicken IgY is not cross-reactive with mammalian IgG. Avian-produced immunoglobulins are potentially more useful in mammalian species than are mammal-produced immunoglobulins, due to the phylogenetic distance between birds and mammals.
IgY has been orally administered successfully for treatment of several gastrointestinal infections, including human rotaviruses, enterotoxigenic Escherichia coli, and other infections caused by Salmonella, Staphyloccocus and Pseudomonas species. It also may be used to convey passive immunity to humans and other animals.
Many prior art methods of imrnunoglobulin isolation involve precipitation and the use of precipitating agents, such as ethanol, polyethylene glycol, lyotropic salts such as copper sulphate, ammonium sulphate and ammonium phosphate, and caprylic acid. The prior art does not teach a commercial scale method of immunoglobulin isolation from egg yolks which does not require such chemical agents, nor an IgY product which is free of the residues of such chemical agents.
While it is known in the art to provide a process for isolation of highly pure IgY from chicken egg yolk in a laboratory setting, the potential uses for IgY require a process for such isolation which is simple, cost-effective and may be used on a commercial scale.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for commercial scale isolation of highly purified immunoglobulin Y from large volumes of chicken egg yolk.
Such a method ideally should only incorporate chemicals permitted for use as a food or other edible product. It is a further object to provide a highly purified IgY
isolate obtained by such a process. Other objects include providing a highly purified IgY isolate for use in edible products, including food, animal feed and dietary supplements.
According to one embodiment of a method of the invention, there is provided a method for extraction and purification of IgY from immune avian egg yolk comprising the following steps: separating the egg yolk from the albumen in a manner which provides a source of egg containing high solids egg yolk with a solids content of between 23% and 50%; diluting the high solids egg yolk obtained from 3-fold to 15-fold in treated water having a dilution temperature between 0.5 and 8 C to obtain diluted egg yolk;
stirring the diluted egg yolk obtained at a stirring temperature of between 0.5 and 8 C until it is mixed to obtain an egg yolk mixture; allowing the egg yolk mixture obtained to settle in a settling container for a settling time of between 16 and 96 hours at a settling temperature of between 0.5 and 8 C, until the mixture separates into a water soluble fraction and a precipitated lipophilic fraction; separating the water soluble fraction and the precipitated lipophilic fraction; concentrating the water soluble fraction through a first membrane filtration system to obtain a filtered material; passing the filtered material through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY;
eluting the adsorbed IgY from the ion exchange adsorption column with an eluting solution to obtain an eluant; filtering the eluant through a second membrane filtration system to obtain a 4%
to 25% IgY concentrate; and drying the IgY concentrate of step (i) to obtain a 40% to 98% IgY isolate.
The solids content of the egg yolk may be at least 44%, preferably at least 46.5% solids.
Each of the dilution, stirring and settling temperatures may be between 3 and 5 C, optimally 4 C. The settling time may be 36 hours. The eluting solution may be a sodium ion containing solution.
According to another embodiment of a method of the invention, there is provided a method for extraction and purification of IgY from immune avian egg yolk comprising the following steps: diluting a mixture of liquid egg yolk and liquid egg white from 3-fold to 15-fold in treated water having a temperature between 0.5 and 8 C;
stirring the diluted mixture obtained at a stirring temperature of between 0.5 and 8 C
until it may be mixed to obtain an egg mixture; separating the egg mixture into a first water soluble fraction, an unseparated mixture fraction, and a first separated lipophilic fraction;
separating the first water soluble fraction; the unseparated mixture fraction, and the first separated lipophilic fraction; centrifuging the unseparated mixture fraction at a centrifugation force of between 1500 and 10,000 g for a centrifugation time of between I
and 120 minutes until it separates into a second water soluble fraction and a second separated lipophilic fraction; adding the second water soluble fraction to the first water soluble fraction to form a combined water soluble fraction; passing the combined water soluble fraction through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY; eluting the adsorbed IgY of from the ion exchange adsorption column with an eluting solution to obtain an eluant; filtering the eluant of through a first membrane filtration system to obtain a 4% to 25% IgY concentrate; and drying the IgY
concentrate to obtain a 40% to 98% IgY isolate.
Each of the dilution, stirring and settling temperatures may be between 3 and 5 C, optimally 4 C. The settling time may be 36 hours. The eluting solution may be a sodium ion containing solution. The centrifugation time may be between 1 and minutes. The centrifugation force may be 3000 g.
The method may include a step after the ion exchange adsorption step of concentrating the water soluble fraction using a second membrane filtration system, and it may include a step after the elution step of concentrating the water soluble fraction using a third membrane filtration system.
The first separated lipophilic fraction may be combined with the second precipitate lipophilic fraction and the resultant combined precipitate lipophilic fraction may be further processed to isolate commercially useful components thereof.
According to a further embodiment of the present invention, there is provided a method for extraction and purification of IgY from immune avian egg yolk comprising the following steps: diluting a mixture of liquid egg yolk and liquid egg white from 3-fold to 15-fold in treated water having a dilution temperature between 0.5 and 8 C;
centrifuging the mixture at a centrifugation force of between 1500 and 10,000 g for a centrifugation time of between 1 and 120 minutes until it separates into a water soluble fraction and a separated lipophilic fraction; separating the water soluble fraction and the precipitated lipophilic fraction; passing the water soluble fraction through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY; eluting the adsorbed IgY
from the ion exchange adsorption column with an eluting solution; filtering the eluant through a first membrane filtration system to obtain a 4% to 25% IgY concentrate; and drying the IgY concentrate to obtain a 40% to 98% IgY isolate.
Each of the dilution, stirring and settling temperatures may be between 3 and 5 C, optimally 4 C. The settling time may be 36 hours. The eluting solution may be a sodium ion containing solution. The centrifugation time may be between 1 and minutes. The centrifugation force may be 3000 g.
The method may include a step after the ion exchange adsorption step of concentrating the water soluble fraction using a second membrane filtration system, and it may include a step after the elution step of concentrating the water soluble fraction using a third membrane filtration system.
According to another embodiment of the present invention, there is provided an IgY
isolate obtained by the methods described herein. The IgY isolate may be a food-safe IgY isolate.
According to another aspect of the present invention, there is provided the use of a food-safe IgY isolate in an edible product.
In another aspect, the invention provides an IgY-rich isolate obtained by the method described herein. In a further aspect, the invention provides a use of the IgY-rich isolate obtained by the method described herein in an edible food product. In still a further aspect, the invention provides a method of treatment of acne comprising administration to a patient having acne a therapeutic amount of IgY-rich product obtained by the process described herein. In another of its aspects, the invention provides a method of passive immunization against pylori bacteria comprising administering to a patient an amount of IgY-rich product obtained by the process described herein sufficient to passively immunize the patient.
The IgY-rich isolate obtained by the method of the present invention does not require extraction additives such as fatty acids or detergents. It is safe for human or animal consumption, that is, it is edible. The IgY-rich isolate is free of any chemical residues.
FIELD OF THE INVENTION
The present invention relates to a method for isolation of egg yolk immunoglobulins. In particular, the present invention relates to a method for isolation of highly purified immunoglobulins from avian egg yolk on a commercial scale.
BACKGROUND OF THE INVENTION
Egg yolk separated from eggs obtained from poultry is an excellent source of polyclonal antibodies. For example, immunoglobulin Y ('IgY') may be isolated from chicken egg yolk.
There are several advantages to obtaining immunoglobulins from chicken egg yolks. Use of egg yolk does not require blood extraction, as is the case with mammalian antibody sources, so it may be easier to comply with animal protection regulations.
Chicken eggs also provide a continual supply of source material. The extent of non-specific binding by IgY is reduced, relative to immunoglobulin sourced from mammals, as chicken IgY is not cross-reactive with mammalian IgG. Avian-produced immunoglobulins are potentially more useful in mammalian species than are mammal-produced immunoglobulins, due to the phylogenetic distance between birds and mammals.
IgY has been orally administered successfully for treatment of several gastrointestinal infections, including human rotaviruses, enterotoxigenic Escherichia coli, and other infections caused by Salmonella, Staphyloccocus and Pseudomonas species. It also may be used to convey passive immunity to humans and other animals.
Many prior art methods of imrnunoglobulin isolation involve precipitation and the use of precipitating agents, such as ethanol, polyethylene glycol, lyotropic salts such as copper sulphate, ammonium sulphate and ammonium phosphate, and caprylic acid. The prior art does not teach a commercial scale method of immunoglobulin isolation from egg yolks which does not require such chemical agents, nor an IgY product which is free of the residues of such chemical agents.
While it is known in the art to provide a process for isolation of highly pure IgY from chicken egg yolk in a laboratory setting, the potential uses for IgY require a process for such isolation which is simple, cost-effective and may be used on a commercial scale.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for commercial scale isolation of highly purified immunoglobulin Y from large volumes of chicken egg yolk.
Such a method ideally should only incorporate chemicals permitted for use as a food or other edible product. It is a further object to provide a highly purified IgY
isolate obtained by such a process. Other objects include providing a highly purified IgY isolate for use in edible products, including food, animal feed and dietary supplements.
According to one embodiment of a method of the invention, there is provided a method for extraction and purification of IgY from immune avian egg yolk comprising the following steps: separating the egg yolk from the albumen in a manner which provides a source of egg containing high solids egg yolk with a solids content of between 23% and 50%; diluting the high solids egg yolk obtained from 3-fold to 15-fold in treated water having a dilution temperature between 0.5 and 8 C to obtain diluted egg yolk;
stirring the diluted egg yolk obtained at a stirring temperature of between 0.5 and 8 C until it is mixed to obtain an egg yolk mixture; allowing the egg yolk mixture obtained to settle in a settling container for a settling time of between 16 and 96 hours at a settling temperature of between 0.5 and 8 C, until the mixture separates into a water soluble fraction and a precipitated lipophilic fraction; separating the water soluble fraction and the precipitated lipophilic fraction; concentrating the water soluble fraction through a first membrane filtration system to obtain a filtered material; passing the filtered material through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY;
eluting the adsorbed IgY from the ion exchange adsorption column with an eluting solution to obtain an eluant; filtering the eluant through a second membrane filtration system to obtain a 4%
to 25% IgY concentrate; and drying the IgY concentrate of step (i) to obtain a 40% to 98% IgY isolate.
The solids content of the egg yolk may be at least 44%, preferably at least 46.5% solids.
Each of the dilution, stirring and settling temperatures may be between 3 and 5 C, optimally 4 C. The settling time may be 36 hours. The eluting solution may be a sodium ion containing solution.
According to another embodiment of a method of the invention, there is provided a method for extraction and purification of IgY from immune avian egg yolk comprising the following steps: diluting a mixture of liquid egg yolk and liquid egg white from 3-fold to 15-fold in treated water having a temperature between 0.5 and 8 C;
stirring the diluted mixture obtained at a stirring temperature of between 0.5 and 8 C
until it may be mixed to obtain an egg mixture; separating the egg mixture into a first water soluble fraction, an unseparated mixture fraction, and a first separated lipophilic fraction;
separating the first water soluble fraction; the unseparated mixture fraction, and the first separated lipophilic fraction; centrifuging the unseparated mixture fraction at a centrifugation force of between 1500 and 10,000 g for a centrifugation time of between I
and 120 minutes until it separates into a second water soluble fraction and a second separated lipophilic fraction; adding the second water soluble fraction to the first water soluble fraction to form a combined water soluble fraction; passing the combined water soluble fraction through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY; eluting the adsorbed IgY of from the ion exchange adsorption column with an eluting solution to obtain an eluant; filtering the eluant of through a first membrane filtration system to obtain a 4% to 25% IgY concentrate; and drying the IgY
concentrate to obtain a 40% to 98% IgY isolate.
Each of the dilution, stirring and settling temperatures may be between 3 and 5 C, optimally 4 C. The settling time may be 36 hours. The eluting solution may be a sodium ion containing solution. The centrifugation time may be between 1 and minutes. The centrifugation force may be 3000 g.
The method may include a step after the ion exchange adsorption step of concentrating the water soluble fraction using a second membrane filtration system, and it may include a step after the elution step of concentrating the water soluble fraction using a third membrane filtration system.
The first separated lipophilic fraction may be combined with the second precipitate lipophilic fraction and the resultant combined precipitate lipophilic fraction may be further processed to isolate commercially useful components thereof.
According to a further embodiment of the present invention, there is provided a method for extraction and purification of IgY from immune avian egg yolk comprising the following steps: diluting a mixture of liquid egg yolk and liquid egg white from 3-fold to 15-fold in treated water having a dilution temperature between 0.5 and 8 C;
centrifuging the mixture at a centrifugation force of between 1500 and 10,000 g for a centrifugation time of between 1 and 120 minutes until it separates into a water soluble fraction and a separated lipophilic fraction; separating the water soluble fraction and the precipitated lipophilic fraction; passing the water soluble fraction through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY; eluting the adsorbed IgY
from the ion exchange adsorption column with an eluting solution; filtering the eluant through a first membrane filtration system to obtain a 4% to 25% IgY concentrate; and drying the IgY concentrate to obtain a 40% to 98% IgY isolate.
Each of the dilution, stirring and settling temperatures may be between 3 and 5 C, optimally 4 C. The settling time may be 36 hours. The eluting solution may be a sodium ion containing solution. The centrifugation time may be between 1 and minutes. The centrifugation force may be 3000 g.
The method may include a step after the ion exchange adsorption step of concentrating the water soluble fraction using a second membrane filtration system, and it may include a step after the elution step of concentrating the water soluble fraction using a third membrane filtration system.
According to another embodiment of the present invention, there is provided an IgY
isolate obtained by the methods described herein. The IgY isolate may be a food-safe IgY isolate.
According to another aspect of the present invention, there is provided the use of a food-safe IgY isolate in an edible product.
In another aspect, the invention provides an IgY-rich isolate obtained by the method described herein. In a further aspect, the invention provides a use of the IgY-rich isolate obtained by the method described herein in an edible food product. In still a further aspect, the invention provides a method of treatment of acne comprising administration to a patient having acne a therapeutic amount of IgY-rich product obtained by the process described herein. In another of its aspects, the invention provides a method of passive immunization against pylori bacteria comprising administering to a patient an amount of IgY-rich product obtained by the process described herein sufficient to passively immunize the patient.
The IgY-rich isolate obtained by the method of the present invention does not require extraction additives such as fatty acids or detergents. It is safe for human or animal consumption, that is, it is edible. The IgY-rich isolate is free of any chemical residues.
BRIEF DESCRIPTION OF THE DRAWING
The invention will be better understood when consideration is given to the following detailed description thereof. Such description makes reference to the annexed drawings wherein:
FIG. 1 is a schematic showing one embodiment of the process of the present invention;
FIG. 2 is a schematic showing an alternate embodiment of the process of the present invention; and FIG. 3 is a schematic showing another embodiment of the process of the present invention.
In the drawings, embodiments of the invention are illustrated by way of example. It is to be expressly understood that the description and drawings are only for the purpose of illustration and as an aid to understanding, and are not intended as a definition of the limits of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
In this specification, the following terms have the meanings set out below:
`avian' means poultry and fowl, including chickens, turkeys, geese, and ducks;
`edible' means not harmful for humans or animals when consumed, and includes food, animal feed, and dietary supplements;
The invention will be better understood when consideration is given to the following detailed description thereof. Such description makes reference to the annexed drawings wherein:
FIG. 1 is a schematic showing one embodiment of the process of the present invention;
FIG. 2 is a schematic showing an alternate embodiment of the process of the present invention; and FIG. 3 is a schematic showing another embodiment of the process of the present invention.
In the drawings, embodiments of the invention are illustrated by way of example. It is to be expressly understood that the description and drawings are only for the purpose of illustration and as an aid to understanding, and are not intended as a definition of the limits of the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
In this specification, the following terms have the meanings set out below:
`avian' means poultry and fowl, including chickens, turkeys, geese, and ducks;
`edible' means not harmful for humans or animals when consumed, and includes food, animal feed, and dietary supplements;
`edible product' includes a pill, capsule, tablet, lozenge or other composition for nutritional use, illness prevention or therapeutic use;
`food-safe IgY isolate' means an IgY isolate having no residues of synthetic solvents, synthetic salts or other synthetic chemical substances added during the process of isolation of the IgY isolate;
`high solids egg yolk' means egg yolk having 44% to 50% solids;
'IgY' includes all immunoglobulins normally found in avian egg yolk;
`expanded bed adsorption chromatography' means the method of fractionation of a protein-containing solution described in patent applications WO 98/08603, WO
02/096215 and WO 04/082397, and is a method of ion exchange adsorption chromatography;
`membrane filtration' includes microfiltration, ultrafiltration, nanofiltration, reverse osmosis or similar filtration systems;
`n-fold dilution' of substance x means dilution of I part substance x with n parts treated water; and `synthetic solvents, synthetic salts or other synthetic chemical substances' includes ammonium sulphate, ammonium phosphate, copper sulphate, caprylic acid and polyethylene, and excludes citric acid, hydrogen chloride, sodium hydroxide, sodium citrate, other sodium ion containing solutions, and resins used in ion exchange adsorption chromatography.
`food-safe IgY isolate' means an IgY isolate having no residues of synthetic solvents, synthetic salts or other synthetic chemical substances added during the process of isolation of the IgY isolate;
`high solids egg yolk' means egg yolk having 44% to 50% solids;
'IgY' includes all immunoglobulins normally found in avian egg yolk;
`expanded bed adsorption chromatography' means the method of fractionation of a protein-containing solution described in patent applications WO 98/08603, WO
02/096215 and WO 04/082397, and is a method of ion exchange adsorption chromatography;
`membrane filtration' includes microfiltration, ultrafiltration, nanofiltration, reverse osmosis or similar filtration systems;
`n-fold dilution' of substance x means dilution of I part substance x with n parts treated water; and `synthetic solvents, synthetic salts or other synthetic chemical substances' includes ammonium sulphate, ammonium phosphate, copper sulphate, caprylic acid and polyethylene, and excludes citric acid, hydrogen chloride, sodium hydroxide, sodium citrate, other sodium ion containing solutions, and resins used in ion exchange adsorption chromatography.
The present invention provides a process for extracting immunoglobulins from avian eggs. In particular, the process of the invention describes a method of isolation of immunoglobulin Y from egg yolk from immunized or non-immunized chickens.
Vaccination and handling of immunized or non-immunized chickens are well known in the art.
In order to optimize the purity of IgY obtained, it is desirable to obtain an egg yolk source material which is high in solids. Pure chicken egg yolk typically contains more than 50% solids. Egg yolks separated individually in a lab may be blotted with an absorbing towel, increasing the solids to as much as 50%. As egg yolk separation from albumen on a commercial scale results in contamination of the egg yolk by the albumen, which has only about 12% solids composition, commercial egg yolk is only required to contain at least 43% solids. Commercial egg yolk may contain as much as 20%
albumen.
Albumen contains overtransferrin, which is indistinguishable from IgY by the isolation process of the present invention. Albumen may also affect yields through adsorption of albumin protein to column beads in the ion exchange adsorption process step described below. To reduce overtransferrin contamination and albumin adsorption, and thus achieve more highly purified IgY, it is important to use a starting material which is as low in albumen as possible, at least 23% to 50% solids, preferably between 44% and 50%
solids, and most preferably at least 46.5% solids and containing significantly less than 20% albumen. High solids egg yolk may be best obtained from fresh eggs using an in-line breaking process.
Vaccination and handling of immunized or non-immunized chickens are well known in the art.
In order to optimize the purity of IgY obtained, it is desirable to obtain an egg yolk source material which is high in solids. Pure chicken egg yolk typically contains more than 50% solids. Egg yolks separated individually in a lab may be blotted with an absorbing towel, increasing the solids to as much as 50%. As egg yolk separation from albumen on a commercial scale results in contamination of the egg yolk by the albumen, which has only about 12% solids composition, commercial egg yolk is only required to contain at least 43% solids. Commercial egg yolk may contain as much as 20%
albumen.
Albumen contains overtransferrin, which is indistinguishable from IgY by the isolation process of the present invention. Albumen may also affect yields through adsorption of albumin protein to column beads in the ion exchange adsorption process step described below. To reduce overtransferrin contamination and albumin adsorption, and thus achieve more highly purified IgY, it is important to use a starting material which is as low in albumen as possible, at least 23% to 50% solids, preferably between 44% and 50%
solids, and most preferably at least 46.5% solids and containing significantly less than 20% albumen. High solids egg yolk may be best obtained from fresh eggs using an in-line breaking process.
The egg yolk source material may be heat-treated. Such heat-treatment may comprise pasteurization.
As indicated schematically in Fig. 1, according to one embodiment of a .settling process of the present invention, one part high solids egg yolk may be mixed with nine parts treated water at a temperature of 0.5 to 8.0 C, preferably 3.0 to 5.0 C, optimally 4.0 C.
Any dilution from 3-fold to 15-fold may be used, but a 9-fold dilution is optimal. The pH
of the mixture may be adjusted if desired. The water may be purified by a reverse osmosis process to deionize and demineralized the water. Other water treatment procedures may instead be used.
The mixture may be stirred to permit mixing and to encourage precipitation of egg solids.
The mixture may be permitted to settle at a temperature of 0.5 to 8.0 C, preferably 3.0 to 5.0 C, optimally 4.0 C, for at least 16 hours and for as long as 96 hours, optimally 36 hours, during which a phase separation into a water soluble fraction ("WSF") rich in IgY
and a precipitated lipophilic fraction ("PLF") occurs.
The WSF and the PLF may be separated by pouring off, siphoning or similar method.
The WSF may be concentrated through the use of membrane filtration according to a process well known in the art, to obtain a 5% to 7% solids fraction.
This 5% to 7% solids fraction may be run through an ion exchange adsorption column, which may be an expanded bed adsorption column, to isolate and purify the IgY.
Ion exchange adsorption technology is known to persons skilled in the art, and is taught, for example in WO 92/00799, WO 92/18237, WO 97/17132, WO 98/33572, WO 00/57982, WO 01/58924. One method of ion exchange adsorption chromatography of the present invention is described in WO 98/08603, WO 02/096215 and WO 04/082397.
The IgY may be eluted from the adsorbent using a sodium ion containing solution or other eluting solution. The resulting eluant is concentrated and purified through a process of membrane filtration to obtain a 4% to 25% IgY concentrate. Finally, the purified and concentrated IgY concentrate is dried to obtain a 40% to 98% pure IgY isolate.
As depicted schematically in Fig. 2, according to an embodiment of a centrifugation process of the present invention, a mixture of liquid egg yolk and liquid egg white may be combined with treated water. Any dilution from 3-fold to 15-fold may be used, but a 9-fold dilution is optimal. Optionally, the pH of the mixture may be adjusted using sodium citrate, hydrogen chloride, citric acid or sodium hydroxide.
The mixture is centrifuged, preferably at a temperature of 0.5 to 8.0 C, more preferably 3.0 to 5.0 C, optimally 4.0 C, during which a phase separation into a WSF
rich in IgY
and a separated lipophilic fraction ("SLF") occurs.
The WSF and the SLF may be separated by pouring off, siphoning or similar method.
Optionally, the WSF may be concentrated by membrane according to a process well known in the art, to obtain a solids fraction.
This solids fraction, may be run through an ion exchange adsorption column, which may be an expanded bed adsorption column, to isolate and purify the IgY. The IgY
may be eluted from the adsorbent using a sodium ion containing solution or equivalent. The resulting eluant optionally may be membrane filtered. The eluant may then be concentrated and purified through a process of membrane filtration to obtain a 4% to 25%
IgY concentrate. The purified and concentrated IgY concentrate may be dried to obtain a 40% to 98% pure IgY isolate.
As depicted schematically in Fig. 3, according to another embodiment of a centrifugation process of the present invention, a mixture of liquid egg yolk and liquid egg white may be combined with treated water. Preferably the mixture may be maintained at a temperature of 0.5 to 8.0 C, more preferably 3.0 to 5.0 C, optimally 4.0 C.
Any dilution from 3-fold to 15-fold may be used, but a 9-fold dilution is optimal.
The pH of the mixture may be adjusted if desired.
The mixture may be stirred to permit mixing and to encourage precipitation of egg solids.
The mixture may be permitted to settle at a temperature of 0.5 to 8.0 C, preferably 3.0 to 5.0 C, optimally 4.0 C, for at least 16 hours and for as long as 96 hours, optimally 36 hours, during which a phase separation into a first WSF rich in IgY, an unseparated mixture fraction ("UMF"), and a first SLF occurs.
The first WSF, the UMF and the first SLF may be separated by pouring off, siphoning or similar method. The UMF may be centrifuged at an RCF of 1500 to 10,000 g, optimally 3000 g, to effect a second phase separation into a second WSF rich in IgY and a second SLF. The second WSF may be removed by pouring off, siphoning or similar method and combined with the first WSF to form a combined WSF. Optionally, the combined WSF
may be concentrated by membrane filtration to obtain a 5% to 7% solids fraction. The second SLF may be combined with the first SLF to form a combined SLF. The combined SLF may be further processed.
This 5% to 7% solids fraction may be run through an ion exchange adsorption column, which may be an expanded bed adsorption column, to isolate and purify the IgY.
The IgY may be eluted from the adsorbent using a sodium ion containing solution or other eluting solution, with residual material in the adsorption bed directed to a drain. The resulting eluant optionally may be membrane filtered, and may then be concentrated and purified through a process of membrane filtration to obtain a 4% to 25% IgY
concentrate.
The purified and concentrated IgY concentrate may be dried to obtain a 40% to 98% pure IgY isolate.
The resulting IgY isolate comprises 40% to 98% avian immunoglobulin. The yield, concentration and purity of the IgY isolate are measurable by standard methods familiar to those skilled in the art. The resulting IgY isolate may be used in an edible product or as an edible product-additive.
It will be appreciated by those skilled in the art that other variations of the preferred embodiment may also be practiced without departing from the scope of the invention.
As indicated schematically in Fig. 1, according to one embodiment of a .settling process of the present invention, one part high solids egg yolk may be mixed with nine parts treated water at a temperature of 0.5 to 8.0 C, preferably 3.0 to 5.0 C, optimally 4.0 C.
Any dilution from 3-fold to 15-fold may be used, but a 9-fold dilution is optimal. The pH
of the mixture may be adjusted if desired. The water may be purified by a reverse osmosis process to deionize and demineralized the water. Other water treatment procedures may instead be used.
The mixture may be stirred to permit mixing and to encourage precipitation of egg solids.
The mixture may be permitted to settle at a temperature of 0.5 to 8.0 C, preferably 3.0 to 5.0 C, optimally 4.0 C, for at least 16 hours and for as long as 96 hours, optimally 36 hours, during which a phase separation into a water soluble fraction ("WSF") rich in IgY
and a precipitated lipophilic fraction ("PLF") occurs.
The WSF and the PLF may be separated by pouring off, siphoning or similar method.
The WSF may be concentrated through the use of membrane filtration according to a process well known in the art, to obtain a 5% to 7% solids fraction.
This 5% to 7% solids fraction may be run through an ion exchange adsorption column, which may be an expanded bed adsorption column, to isolate and purify the IgY.
Ion exchange adsorption technology is known to persons skilled in the art, and is taught, for example in WO 92/00799, WO 92/18237, WO 97/17132, WO 98/33572, WO 00/57982, WO 01/58924. One method of ion exchange adsorption chromatography of the present invention is described in WO 98/08603, WO 02/096215 and WO 04/082397.
The IgY may be eluted from the adsorbent using a sodium ion containing solution or other eluting solution. The resulting eluant is concentrated and purified through a process of membrane filtration to obtain a 4% to 25% IgY concentrate. Finally, the purified and concentrated IgY concentrate is dried to obtain a 40% to 98% pure IgY isolate.
As depicted schematically in Fig. 2, according to an embodiment of a centrifugation process of the present invention, a mixture of liquid egg yolk and liquid egg white may be combined with treated water. Any dilution from 3-fold to 15-fold may be used, but a 9-fold dilution is optimal. Optionally, the pH of the mixture may be adjusted using sodium citrate, hydrogen chloride, citric acid or sodium hydroxide.
The mixture is centrifuged, preferably at a temperature of 0.5 to 8.0 C, more preferably 3.0 to 5.0 C, optimally 4.0 C, during which a phase separation into a WSF
rich in IgY
and a separated lipophilic fraction ("SLF") occurs.
The WSF and the SLF may be separated by pouring off, siphoning or similar method.
Optionally, the WSF may be concentrated by membrane according to a process well known in the art, to obtain a solids fraction.
This solids fraction, may be run through an ion exchange adsorption column, which may be an expanded bed adsorption column, to isolate and purify the IgY. The IgY
may be eluted from the adsorbent using a sodium ion containing solution or equivalent. The resulting eluant optionally may be membrane filtered. The eluant may then be concentrated and purified through a process of membrane filtration to obtain a 4% to 25%
IgY concentrate. The purified and concentrated IgY concentrate may be dried to obtain a 40% to 98% pure IgY isolate.
As depicted schematically in Fig. 3, according to another embodiment of a centrifugation process of the present invention, a mixture of liquid egg yolk and liquid egg white may be combined with treated water. Preferably the mixture may be maintained at a temperature of 0.5 to 8.0 C, more preferably 3.0 to 5.0 C, optimally 4.0 C.
Any dilution from 3-fold to 15-fold may be used, but a 9-fold dilution is optimal.
The pH of the mixture may be adjusted if desired.
The mixture may be stirred to permit mixing and to encourage precipitation of egg solids.
The mixture may be permitted to settle at a temperature of 0.5 to 8.0 C, preferably 3.0 to 5.0 C, optimally 4.0 C, for at least 16 hours and for as long as 96 hours, optimally 36 hours, during which a phase separation into a first WSF rich in IgY, an unseparated mixture fraction ("UMF"), and a first SLF occurs.
The first WSF, the UMF and the first SLF may be separated by pouring off, siphoning or similar method. The UMF may be centrifuged at an RCF of 1500 to 10,000 g, optimally 3000 g, to effect a second phase separation into a second WSF rich in IgY and a second SLF. The second WSF may be removed by pouring off, siphoning or similar method and combined with the first WSF to form a combined WSF. Optionally, the combined WSF
may be concentrated by membrane filtration to obtain a 5% to 7% solids fraction. The second SLF may be combined with the first SLF to form a combined SLF. The combined SLF may be further processed.
This 5% to 7% solids fraction may be run through an ion exchange adsorption column, which may be an expanded bed adsorption column, to isolate and purify the IgY.
The IgY may be eluted from the adsorbent using a sodium ion containing solution or other eluting solution, with residual material in the adsorption bed directed to a drain. The resulting eluant optionally may be membrane filtered, and may then be concentrated and purified through a process of membrane filtration to obtain a 4% to 25% IgY
concentrate.
The purified and concentrated IgY concentrate may be dried to obtain a 40% to 98% pure IgY isolate.
The resulting IgY isolate comprises 40% to 98% avian immunoglobulin. The yield, concentration and purity of the IgY isolate are measurable by standard methods familiar to those skilled in the art. The resulting IgY isolate may be used in an edible product or as an edible product-additive.
It will be appreciated by those skilled in the art that other variations of the preferred embodiment may also be practiced without departing from the scope of the invention.
Claims (33)
1. A method for extraction and purification of IgY from immune avian egg yolk comprising the following steps:
(a) separating the egg yolk from the albumen in a manner which provides a source of egg containing high solids egg yolk with a solids content of between 23% and 50%;
(b) diluting the high solids egg yolk obtained in step (a) from 3-fold to 15-fold in treated water having a dilution temperature between 0.5 and 8 °C to obtain diluted egg yolk;
(c) stirring the diluted egg yolk obtained from step (b) at a stirring temperature of between 0.5 and 8 °C until it is mixed to obtain an egg yolk mixture;
(d) allowing the egg yolk mixture obtained from step (c) to settle in a settling container for a settling time of between 16 and 96 hours at a settling temperature of between 0.5 and 8 °C, until the mixture separates into a water soluble fraction and a precipitated lipophilic fraction;
(e) separating the water soluble fraction and the precipitated lipophilic fraction from step (d);
(f) concentrating the water soluble fraction of step (e) through a first membrane filtration system to obtain a filtered material;
(g) passing the filtered material of step (f) through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY;
(h) eluting the adsorbed IgY of step (g) from the ion exchange adsorption column with an eluting solution to obtain an eluant;
(i) filtering the eluant of step (h) through a second membrane filtration system to obtain a 4% to 25% IgY concentrate; and (j) drying the IgY concentrate of step (i) to obtain a 40% to 98% IgY isolate.
(a) separating the egg yolk from the albumen in a manner which provides a source of egg containing high solids egg yolk with a solids content of between 23% and 50%;
(b) diluting the high solids egg yolk obtained in step (a) from 3-fold to 15-fold in treated water having a dilution temperature between 0.5 and 8 °C to obtain diluted egg yolk;
(c) stirring the diluted egg yolk obtained from step (b) at a stirring temperature of between 0.5 and 8 °C until it is mixed to obtain an egg yolk mixture;
(d) allowing the egg yolk mixture obtained from step (c) to settle in a settling container for a settling time of between 16 and 96 hours at a settling temperature of between 0.5 and 8 °C, until the mixture separates into a water soluble fraction and a precipitated lipophilic fraction;
(e) separating the water soluble fraction and the precipitated lipophilic fraction from step (d);
(f) concentrating the water soluble fraction of step (e) through a first membrane filtration system to obtain a filtered material;
(g) passing the filtered material of step (f) through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY;
(h) eluting the adsorbed IgY of step (g) from the ion exchange adsorption column with an eluting solution to obtain an eluant;
(i) filtering the eluant of step (h) through a second membrane filtration system to obtain a 4% to 25% IgY concentrate; and (j) drying the IgY concentrate of step (i) to obtain a 40% to 98% IgY isolate.
2. The method of claim 1 wherein the solids content of the egg yolk is at least 44%.
3. The method of claim 2 wherein the solids content of the egg yolk is at least 46.5% solids.
4. The method of claim 1, wherein each of the dilution, stirring and settling temperatures is between 3 and 5 °C.
5. The method of claim 3, wherein each of the dilution, stirring and settling temperatures is 4 °C.
6. The method of claim 1, wherein the settling time is 36 hours.
7. The method of claim 1, wherein the eluting solution is a sodium ion containing solution.
8. A method for extraction and purification of IgY from immune avian egg yolk comprising the following steps:
(a) diluting a mixture of liquid egg yolk and liquid egg white from 3-fold to 15-fold in treated water having a temperature between 0.5 and 8°C;
(b) stirring the diluted mixture obtained from step (a) at a stirring temperature of between 0.5 and 8 °C until it is mixed to obtain an egg mixture;
(c) separating the egg mixture of step (b) into a first water soluble fraction, an unseparated mixture fraction, and a first separated lipophilic fraction;
(d) separating the first water soluble fraction; the unseparated mixture fraction, and the first separated lipophilic fraction;
(e) centrifuging the unseparated mixture fraction at a centrifugation force of between 1500 and 10,000 g for a centrifugation time of between 1 and 120 minutes until it separates into a second water soluble fraction and a second separated lipophilic fraction;
(f) adding the second water soluble fraction to the first water soluble fraction to form a combined water soluble fraction;
(g) passing the combined water soluble fraction of step (f) through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY;
(h) eluting the adsorbed IgY of step (g) from the ion exchange adsorption column with an eluting solution to obtain an eluant;
(i) filtering the eluant of step (h) through a first membrane filtration system to obtain a 4% to 25% IgY concentrate;
(j) drying the IgY concentrate of step (i) to obtain a 40% to 98% IgY isolate.
(a) diluting a mixture of liquid egg yolk and liquid egg white from 3-fold to 15-fold in treated water having a temperature between 0.5 and 8°C;
(b) stirring the diluted mixture obtained from step (a) at a stirring temperature of between 0.5 and 8 °C until it is mixed to obtain an egg mixture;
(c) separating the egg mixture of step (b) into a first water soluble fraction, an unseparated mixture fraction, and a first separated lipophilic fraction;
(d) separating the first water soluble fraction; the unseparated mixture fraction, and the first separated lipophilic fraction;
(e) centrifuging the unseparated mixture fraction at a centrifugation force of between 1500 and 10,000 g for a centrifugation time of between 1 and 120 minutes until it separates into a second water soluble fraction and a second separated lipophilic fraction;
(f) adding the second water soluble fraction to the first water soluble fraction to form a combined water soluble fraction;
(g) passing the combined water soluble fraction of step (f) through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY;
(h) eluting the adsorbed IgY of step (g) from the ion exchange adsorption column with an eluting solution to obtain an eluant;
(i) filtering the eluant of step (h) through a first membrane filtration system to obtain a 4% to 25% IgY concentrate;
(j) drying the IgY concentrate of step (i) to obtain a 40% to 98% IgY isolate.
9. The method of claim 8, wherein each of the dilution, stirring and settling temperatures is between 3 and 5 °C.
10. The method of claim 9, wherein each of the dilution, stirring and settling temperatures is 4 °C.
11. The method of claim 8, wherein the centrifugation time is between 1 and 15 minutes.
12. The method of claim 8, wherein the centrifugation force is 3000 g.
13. The method of claim 8 further comprising a step after the ion exchange adsorption step of concentrating the water soluble fraction using a second membrane filtration system.
14. The method of claim 8, wherein the eluting solution is a sodium ion containing solution.
15. The method of claim 8 further comprising a step after the elution step of concentrating the water soluble fraction using a third membrane filtration system.
16. The method of claim 8, wherein the first separated lipophilic fraction of step (c) is combined with the second precipitate lipophilic fraction of step (e) and the resultant combined precipitate lipophilic fraction is further processed to isolate commercially useful components thereof.
17. A method for extraction and purification of IgY from immune avian egg yolk comprising the following steps:
(a) diluting a mixture of liquid egg yolk and liquid egg white from 3-fold to 15-fold in treated water having a dilution temperature between 0.5 and 8°C;
(b) centrifuging the mixture of step (a) at a centrifugation force of between 1500 and 10,000 g for a centrifugation time of between 1 and 120 minutes until it separates into a water soluble fraction and a separated lipophilic fraction;
(c) separating the water soluble fraction and the precipitated lipophilic fraction from step (b);
(d) passing the water soluble fraction of step (c) through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY;
(e) eluting the adsorbed IgY of step (d) from the ion exchange adsorption column with an eluting solution;
(f) filtering the eluant of step (e) through a first membrane filtration system to obtain a 4% to 25% IgY concentrate;
(g) drying the IgY concentrate of step (f) to obtain a 40% to 98% IgY isolate.
(a) diluting a mixture of liquid egg yolk and liquid egg white from 3-fold to 15-fold in treated water having a dilution temperature between 0.5 and 8°C;
(b) centrifuging the mixture of step (a) at a centrifugation force of between 1500 and 10,000 g for a centrifugation time of between 1 and 120 minutes until it separates into a water soluble fraction and a separated lipophilic fraction;
(c) separating the water soluble fraction and the precipitated lipophilic fraction from step (b);
(d) passing the water soluble fraction of step (c) through an ion exchange adsorption column containing IgY-adsorbent beads to adsorb IgY;
(e) eluting the adsorbed IgY of step (d) from the ion exchange adsorption column with an eluting solution;
(f) filtering the eluant of step (e) through a first membrane filtration system to obtain a 4% to 25% IgY concentrate;
(g) drying the IgY concentrate of step (f) to obtain a 40% to 98% IgY isolate.
18. The method of claim 17, wherein the dilution temperature is between 3 and 5°C.
19. The method of claim 18, wherein the dilution temperature is 4°C.
20. The method of claim 17, wherein the centrifugation time is between 1 and minutes.
21. The method of claim 17, wherein the centrifugation force is 3000 g.
22. The method of claim 17 further comprising a step (c1) after step (c) of concentrating the water soluble fraction using a second membrane filtration system.
23. The method of claim 17, wherein the eluting solution is a sodium ion containing solution.
24. The method of claim 17 further comprising a step (e1) after step (e) of concentrating the water soluble fraction using a third membrane filtration system.
25. An IgY isolate obtained by the method of claim 1.
26. The IgY isolate of claim 25, wherein the IgY isolate is a food-safe IgY
isolate.
isolate.
27. The use of the food-safe IgY isolate of claim 26 in an edible product.
28. An IgY isolate obtained by the method of claim 8.
29. The IgY isolate of claim 28, wherein the IgY isolate is a food-safe IgY
isolate.
isolate.
30. The use of the food-safe IgY isolate of claim 29 in an edible product.
31. An IgY isolate obtained by the method of claim 17.
32. The IgY isolate of claim 31, wherein the IgY isolate is a food-safe IgY
isolate.
isolate.
33. The use of the food-safe IgY isolate of claim 32 in an edible product.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US88585607P | 2007-01-19 | 2007-01-19 | |
| US60/885,856 | 2007-01-19 | ||
| PCT/CA2008/000100 WO2008086621A1 (en) | 2007-01-19 | 2008-01-18 | A method for commercial isolation of egg yolk igy, its product, and uses of the product |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2712375A1 true CA2712375A1 (en) | 2008-07-24 |
Family
ID=39635610
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2712375A Abandoned CA2712375A1 (en) | 2007-01-19 | 2008-01-18 | A method for commercial isolation of egg yolk igy, its product, and uses of the product |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA2712375A1 (en) |
| WO (1) | WO2008086621A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8652457B2 (en) | 2012-07-13 | 2014-02-18 | Wisconsin Alumni Research Foundation | Interleukin-10 peptides and antibodies thereof for inhibiting adverse effects of protozoan infection |
| US20150037277A1 (en) | 2013-08-02 | 2015-02-05 | The United States Of America, As Represented By The Secretary Of Agriculture | Methods of reducing salmonella in poultry |
| US9821028B2 (en) | 2014-07-11 | 2017-11-21 | Wisconsin Alumni Research Foundation | Methods of controlling parasitic worms in animals |
| US20160280778A1 (en) | 2015-03-27 | 2016-09-29 | Wisconsin Alumni Research Foundation | Compositions and methods of use of interleukin-10 peptides and anti-interleukin-10 antibodies |
| US10881129B2 (en) | 2017-08-10 | 2021-01-05 | Wisconsin Alumni Research Foundation | Coated egg yolk cores, methods of making and methods of use thereof |
| EP3681910B1 (en) | 2017-09-12 | 2023-01-25 | Wisconsin Alumni Research Foundation | Interleukin-10 receptor-2 peptides, antibodies, compositions, and methods of use thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2323571T3 (en) * | 2002-06-11 | 2009-07-21 | Good Biotech Corporation | PROCESS FOR SELECTLY INSULATING AN IGEMA YEG EGG BODIES FROM AN ANSERIFORM BIRD. |
-
2008
- 2008-01-18 WO PCT/CA2008/000100 patent/WO2008086621A1/en active Application Filing
- 2008-01-18 CA CA2712375A patent/CA2712375A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| WO2008086621A1 (en) | 2008-07-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| De Meulenaer et al. | Isolation and purification of chicken egg yolk immunoglobulins: a review | |
| US5367054A (en) | Large-scale purification of egg immunoglobulin | |
| KR101645957B1 (en) | Process for producing milk fractions rich in secretory immunoglobulins | |
| CA2712375A1 (en) | A method for commercial isolation of egg yolk igy, its product, and uses of the product | |
| US8080272B2 (en) | Method of separating components of technical eggs, edible eggs, yolk and whites and products therefrom | |
| EP0503293B1 (en) | Specific chicken egg antibody and method for its production | |
| Lee et al. | Sequential separation of immunoglobulin Y and phosvitin from chicken egg yolk without using organic solvents | |
| US20110015374A1 (en) | Method for Commercial Isolation of Egg Yolk IgY, Its Product, and Uses of the Product | |
| JP4316012B2 (en) | Egg anti-inflammatory composition, isolation method and use | |
| Bizanov | IgY extraction and purification from chicken egg yolk | |
| RU2366294C1 (en) | Dietary supplement "mobelis" and its production method | |
| CN114014926A (en) | Method for simply and rapidly preparing high-purity immunoglobulin G1 and G2 from bovine colostrum | |
| RU2400106C1 (en) | Method for production of "l-pfi" dietary supplement from secondary milk stock and "l-pfi" dietary supplement produced by this method | |
| US5747031A (en) | Process for isolating immunoglobulins in whey | |
| KR20150081789A (en) | Sequential separation of lysozyme, ovomucin, ovotransferrin and ovalbumin from egg white | |
| WO2020058936A1 (en) | Process for purifying an antibody from egg yolk, products and uses thereof | |
| JP2002535279A (en) | Egg anti-inflammatory composition and method of treating and preventing inflammation | |
| Hoenig et al. | Suppression of humoral immune response in mice by administration of high molecular levan | |
| RU2084229C1 (en) | Method of preparing immunoglobulin preparation for prophylaxis and therapy of bacterial and viral diseases | |
| NZ510871A (en) | Method of processing a proteinous material, a product so obtained, and use thereof | |
| Ebrahimi et al. | Glycomacropeptide: A comprehensive understanding of its major biological characteristics and purification methodologies | |
| Mulvihill et al. | Production of whey-protein-enriched products | |
| US20040182785A1 (en) | Animal plasma process by precipitation | |
| AU2019252178B2 (en) | Method for separation of proteins naturally occurring in mammalian milk | |
| US20210259282A1 (en) | Process for the purification of whey protein isolate and formulation thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |
Effective date: 20140120 |