Method for separating lysozyme from egg-white

Claim 1

A method for separating lysozyme from egg-white which has a lysozyme fraction and an albumin fraction, without destroying the commercial utility of the lysozyme fraction or the albumin fraction, comprising the steps of: (a) preferentially, reversibly binding the lysozyme fraction to an affinity resin at about the natural pH of the egg-white by contacting the egg-white with the resin; (b) separating the albumin fraction from the resin to leave the lysozyme fraction bound to the resin and to obtain an albumin fraction of commercial value; and (c) eluting the bound lysozyme from the resin to obtain a lysozyme solution and a reusable resin.

Claim 2

A method for separating lysozyme from egg-white which has a lysozyme fraction and an albumin fraction, without destroying the commercial utility of the lysozyme fraction or the albumin fraction, comprising the steps of: (a) preferentially, reversibly binding the lysozyme fraction to an affinity resin at about the natural pH of the egg-white by contacting the egg-white with the resin; (b) separating the albumin fraction from the resin to leave the lysozyme fraction bound to the resin and to obtain an albumin fraction of commercial value; and (c) eluting the bound lysozyme from the resin to obtain a lysozyme solution and a reusable resin.

Claim 3

A method for separating lysozyme from egg-white which has a lysozyme fraction and an albumin fraction, without destroying the commercial utility of the lysozyme fraction or the albumin fraction, comprising the steps of: (a) charging the egg-white to a stirred reactor; (b) preferentially, reversibly binding the lysozyme fraction to an affinity resin by immersing the resin within the egg-white at about the natural pH of the egg-white; (c) filtering the mixture of resin and egg-white to separate the resin from the albumin fraction; (d) removing the albumin fraction from the resin to leave the lysozyme bound to the resin and to obtain an albumin fraction of commercial value; and (e) eluting the bound lysozyme from the resin with a salt solution to obtain a lysozyme solution and a reusable resin.

Claim 4

A method for separating lysozyme from egg-white which has a lysozyme fraction and an albumin fraction, without destroying the commercial utility of the lysozyme fraction or the albumin fraction, comprising the steps of: (a) charging the egg-white to a stirred reactor; (b) preferentially, reversibly binding the lysozyme fraction to an affinity resin by immersing the resin within the egg-white at about the natural pH of the egg-white; (c) filtering the mixture of resin and egg-white to separate the resin from the albumin fraction; (d) removing the albumin fraction from the resin to leave the lysozyme bound to the resin and to obtain an albumin fraction of commercial value; and (e) eluting the bound lysozyme from the resin with a salt solution to obtain a lysozyme solution and a reusable resin.

Claim 5

A method for separating lysozyme from egg-white which has a lysozyme fraction and an albumin fraction, without destroying the commercial utility of the lysozyme fraction or the albumin fraction, comprising the steps of: (a) charging the egg-white to a stirred reactor; (b) preferentially, reversibly binding the lysozyme fraction to an affinity resin at about the natural pH of the egg-white by immersing and mixing the resin within the egg-white; (c) separating the albumin fraction from the resin to leave the lysozyme fraction bound to the resin and to obtain an albumin fraction of commercial value; (d) washing the resin to leave the lysozyme bound, but to remove undesired proteins from the resin; and (e) eluting the bound lysozyme from the resin with a salt solution to obtain a lysozyme fraction and a reusable resin.

Claim 6

A method for separating lysozyme from egg-white which has a lysozyme fraction and an albumin fraction, without destroying the commercial utility of the lysozyme fraction or the albumin fraction, comprising the steps of: (a) charging the egg-white to a stirred reactor; (b) preferentially, reversibly binding the lysozyme fraction to an affinity resin at about the natural pH of the egg-white by immersing and mixing the resin within the egg-white; (c) separating the albumin fraction from the resin to leave the lysozyme fraction bound to the resin and to obtain an albumin fraction of commercial value; (d) washing the resin to leave the lysozyme bound, but to remove undesired proteins from the resin; and (e) eluting the bound lysozyme from the resin with a salt solution to obtain a lysozyme fraction and a reusable resin.

Claim 7

A method for separating lysozyme from egg-white which has a lysozyme fraction and an albumin fraction, without destroying the commercial utility of the lysozyme fraction or the albumin fraction, comprising the steps of: (a) placing an affinity resin in a packed column; (b) passing egg-whites through the column to preferentially, reversibly bind the lysozyme fraction to the resin by chemical affinity; (c) separating the resin from the albumin fraction of the egg-white, leaving the lysozyme bound to the resin and obtaining a commercially valuable albumin fraction; and (d) eluting the bound lysozyme from the resin with a salt solution to obtain a lysozyme fraction and a reusable resin.

Claim 8

A method for separating lysozyme from egg-white which has a lysozyme fraction and an albumin fraction, without destroying the commercial utility of the lysozyme fraction or the albumin fraction, comprising the steps of: (a) placing an affinity resin in a packed column; (b) passing egg-whites through the column to preferentially, reversibly bind the lysozyme fraction to the resin by chemical affinity; (c) separating the resin from the albumin fraction of the egg-white, leaving the lysozyme bound to the resin and obtaining a commercially valuable albumin fraction; and (d) eluting the bound lysozyme from the resin with a salt solution to obtain a lysozyme fraction and a reusable resin.

Claim 18

A method for separating lysozyme from egg-white which has a lysozyme fraction and an albumin fraction, without destroying the commercial utility of the lysozyme fraction or the albumin fraction, comprising the steps of: (a) preferentially, reversibly binding the lysozyme fraction to a kappa-carrageenan-chitosan affinity resin at about the natural pH of the egg-white by contacting the egg-white with the resin; (b) separating the albumin fraction from the resin to leave the lysozyme fraction bound to the resin and to obtain an albumin fraction of commercial value; and (c) eluting the bound lysozyme from the resin to obtain a lysozyme solution and a reusable resin.

Claim 19

A method for separating lysozyme from egg-white which has a lysozyme fraction and an albumin fraction, without destroying the commercial utility of the lysozyme fraction or the albumin fraction, comprising the steps of: (a) preferentially, reversibly binding the lysozyme fraction to a kappa-carrageenan-chitosan affinity resin at about the natural pH of the egg-white by contacting the egg-white with the resin; (b) separating the albumin fraction from the resin to leave the lysozyme fraction bound to the resin and to obtain an albumin fraction of commercial value; and (c) eluting the bound lysozyme from the resin to obtain a lysozyme solution and a reusable resin.

Claim 26

A method for separating lysozyme from egg-white which has a lysozyme fraction and an albumin fraction, comprising the steps of: (a) chilling the egg-white; (b) preferentially binding the lysozyme fraction to an affinity resin by contacting the egg-white with the resin at about the natural pH of the egg-white while maintaining the temperature below about 10° C.; (c) separating the albumin fraction from the resin to leave the lysozyme fraction bound to the resin and to obtain a commercially valuable albumin fraction; (d) washing the resin to remove unwanted proteins from the resin while leaving the lysozyme bound to the resin; (e) eluting the lysozyme from the resin by washing the resin with an aqueous KCl solution having an ion concentration of at least about 1M; (f) crystallizing the lysozyme by increasing the salt concentration to about 0.7-1.5M KCl, adjusting the pH to about the isoelectric point, and cooling the solution to about 5° C.; (g) filtering the chilled solution to recover crystalline lysozyme; (h) redissolving the lysozyme to form a solution of between about 7-15% lysozyme by weight; (i) filtering the solution to remove denatured and contaminating proteins; (j) dialyzing the filtered solution at a pH of less than 5.0 to isolate lysozyme chloride; (k) filtering the lysozyme chloride to obtain a filtered product solution; and (l) drying the filtered product solution to obtain powdered lysozyme chloride.

Claim 27

A method for separating lysozyme from egg-white which has a lysozyme fraction and an albumin fraction, comprising the steps of: (a) chilling the egg-white; (b) preferentially binding the lysozyme fraction to an affinity resin by contacting the egg-white with the resin at about the natural pH of the egg-white while maintaining the temperature below about 10° C.; (c) separating the albumin fraction from the resin to leave the lysozyme fraction bound to the resin and to obtain a commercially valuable albumin fraction; (d) washing the resin to remove unwanted proteins from the resin while leaving the lysozyme bound to the resin; (e) eluting the lysozyme from the resin by washing the resin with an aqueous KCl solution having an ion concentration of at least about 1M; (f) crystallizing the lysozyme by increasing the salt concentration to about 0.7-1.5M KCl, adjusting the pH to about the isoelectric point, and cooling the solution to about 5° C.; (g) filtering the chilled solution to recover crystalline lysozyme; (h) redissolving the lysozyme to form a solution of between about 7-15% lysozyme by weight; (i) filtering the solution to remove denatured and contaminating proteins; (j) dialyzing the filtered solution at a pH of less than 5.0 to isolate lysozyme chloride; (k) filtering the lysozyme chloride to obtain a filtered product solution; and (l) drying the filtered product solution to obtain powdered lysozyme chloride.

Claim 33

A method for separating lysozyme from hen egg-white which has a lysozyme fraction and an albumin fraction, without destroying the commercial utility of the lysozyme fraction and the albumin fraction, comprising the steps of: (a) chilling the hen egg-white; (b) preferentially binding the lysozyme fraction to an affinity resin selected from the group consisting of: agar; agarose; kappa-carrageenan; chitosan; agarose--kappa-carrageenan; agarose-chitosan; kappa-carrageenan--chitosan; agarose--kappa-carrageenan--chitosan; agar--kappa-carrageenan--chitosan; agar--kappa-carrageenan; and mixtures thereof by contacting the hen egg-white with the resin at about the natural pH of the hen egg-white and at a temperature below about 10° C. for between about 15-60 minutes until the resin is substantially saturated with proteins; (c) separating the albumin fraction from the resin to leave the lysozyme fraction bound to the resin; (d) washing the resin with water to remove unwanted proteins from the resin while leaving the lysozyme bound to the resin; (e) eluting the lysozyme from the resin by washing the resin with an aqueous KCl solution having an ion concentration of at least about 1M to leave a reusable resin; (f) crystallizing the lysozyme by increasing the salt concentration to about 0.7-1.5M KCl, adjusting the pH to about the isoelectric point, and cooling the solution to about 5° C.; (g) filtering the chilled solution to recover raw, crystalline lysozyme; (h) redissolving the raw lysozyme to form a solution of between about 7-15% lysozyme by weight; (i) filtering the solution to remove denatured and contaminating proteins; (j) dialyzing the filtered solution at a pH of between about 3.0 to 4.0 and at a temperature no greater than about 5° C. to isolate lysozyme chloride; (k) filtering the lysozyme chloride to obtain a filtered product solution; and (l) drying the filtered product solution to obtain powdered lysozyme chloride.

Claim 34

A method for separating lysozyme from hen egg-white which has a lysozyme fraction and an albumin fraction, without destroying the commercial utility of the lysozyme fraction and the albumin fraction, comprising the steps of: (a) chilling the hen egg-white; (b) preferentially binding the lysozyme fraction to an affinity resin selected from the group consisting of: agar; agarose; kappa-carrageenan; chitosan; agarose--kappa-carrageenan; agarose-chitosan; kappa-carrageenan--chitosan; agarose--kappa-carrageenan--chitosan; agar--kappa-carrageenan--chitosan; agar--kappa-carrageenan; and mixtures thereof by contacting the hen egg-white with the resin at about the natural pH of the hen egg-white and at a temperature below about 10° C. for between about 15-60 minutes until the resin is substantially saturated with proteins; (c) separating the albumin fraction from the resin to leave the lysozyme fraction bound to the resin; (d) washing the resin with water to remove unwanted proteins from the resin while leaving the lysozyme bound to the resin; (e) eluting the lysozyme from the resin by washing the resin with an aqueous KCl solution having an ion concentration of at least about 1M to leave a reusable resin; (f) crystallizing the lysozyme by increasing the salt concentration to about 0.7-1.5M KCl, adjusting the pH to about the isoelectric point, and cooling the solution to about 5° C.; (g) filtering the chilled solution to recover raw, crystalline lysozyme; (h) redissolving the raw lysozyme to form a solution of between about 7-15% lysozyme by weight; (i) filtering the solution to remove denatured and contaminating proteins; (j) dialyzing the filtered solution at a pH of between about 3.0 to 4.0 and at a temperature no greater than about 5° C. to isolate lysozyme chloride; (k) filtering the lysozyme chloride to obtain a filtered product solution; and (l) drying the filtered product solution to obtain powdered lysozyme chloride.

Claim 37

A method for separating lysozyme from substantially undiluted hen egg-white which has a lysozyme fraction and an albumin fraction, comprising the steps of: (a) chilling the egg-white; (b) preferentially binding the lysozyme fraction to an affinity resin by contacting the egg-white with the resin at about the natural pH of the egg-white while maintaining the temperature below about 10° C.; (c) separating the albumin fraction from the resin to leave the lysozyme fraction bound to the resin and to obtain a commercially valuable albumin fraction; (d) washing the resin to remove unwanted proteins from the resin while leaving the lysozyme bound to the resin; (e) eluting the lysozyme from the resin by washing the resin with an aqueous KCl solution having an ion concentration of at least about 1M; (f) crystallizing the lysozyme by increasing the salt concentration to about 0.7-1.5M KCl, adjusting the pH to about the isoelectric point, and cooling the solution to about 5° C.; (g) filtering the chilled solution to recover crystalline lysozyme; (h) redissolving the lysozyme to form a solution of between about 7-15% lysozyme by weight; (i) filtering the solution to remove denatured and contaminating proteins; and (j) purifying the filtered solution to obtain a commercial product of the lysozyme or of a salt of the lysozyme.

Claim 38

A method for separating lysozyme from substantially undiluted hen egg-white which has a lysozyme fraction and an albumin fraction, comprising the steps of: (a) chilling the egg-white; (b) preferentially binding the lysozyme fraction to an affinity resin by contacting the egg-white with the resin at about the natural pH of the egg-white while maintaining the temperature below about 10° C.; (c) separating the albumin fraction from the resin to leave the lysozyme fraction bound to the resin and to obtain a commercially valuable albumin fraction; (d) washing the resin to remove unwanted proteins from the resin while leaving the lysozyme bound to the resin; (e) eluting the lysozyme from the resin by washing the resin with an aqueous KCl solution having an ion concentration of at least about 1M; (f) crystallizing the lysozyme by increasing the salt concentration to about 0.7-1.5M KCl, adjusting the pH to about the isoelectric point, and cooling the solution to about 5° C.; (g) filtering the chilled solution to recover crystalline lysozyme; (h) redissolving the lysozyme to form a solution of between about 7-15% lysozyme by weight; (i) filtering the solution to remove denatured and contaminating proteins; and (j) purifying the filtered solution to obtain a commercial product of the lysozyme or of a salt of the lysozyme.