Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Albumin
Reexamination Certificate
1999-04-30
2003-03-04
Saunders, David (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Albumin
C210S669000, C210S691000, C426S271000, C530S366000, C530S387100, C530S395000, C530S414000, C530S416000, C530S833000
Reexamination Certificate
active
06528622
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a preparative method for separating and recovering whey proteins from a protein solution. More particularly, it relates to a process for separating and recovering proteins from a whey protein solution using ion exchange.
BACKGROUND OF THE INVENTION
It is known in the art that proteins can be fractionated and recovered from whey protein solutions using ion exchange. It is also known that there are a large number of parameters which determine the protein capacity of the ion exchanger, the yield of protein and which particular proteins are adsorbed by which type of ion exchangers under what conditions. For example, the pH of the protein solution and the isoelectric point (IEP) of the protein largely determine whether the protein will bind to a cation or anion exchanger. In addition, the protein is in competition for binding sites with other ionic species in solution, such as salts, and this competition can reduce or even prevent the adsorption of the protein. There are also behavioural differences between the many ion exchangers available for use. The information published over the last twenty years concerning the separation of whey proteins has largely been determined empirically.
It is also known, and the applicants have confirmed, that preconcentration of whey protein solution above a concentration of about 2% protein prior to ion exchange results in a loss of protein capacity in the ion exchanger. This is true for proteins in general. R K S Scopes (in Protein Purification, Principles and Practices, 2nd Edition, pages 101 and 118, Springer-Verlag, N.Y., 1987) advocates the use of protein concentrations of 0.5% and states that 3% is too high especially if a substantial proportion of the protein is going to be adsorbed.
The prior art therefore generally teaches the application of dilute whey protein solutions to ion exchangers. See for example, Palmer (
Process Biochemistry
, 1977, 12, 24-28), Phillips, Jones and Palmer (U.S Pat. No. 4,218,490), Bottomley (EP 0,320,152), Mirabel (GB 1,563,990), Skudder (
J. Daily Research
, 1985, 52, 167-181), Kawasaki and Dosako (EP 0,488,589), Burton and Shudder (GB 2,188,52) and Kawasaki, Dosako, Shimatani and Idota (GB 2,251,858). These references teach the use of whey with a protein concentration of 0.5-0.7% as starting material for contacting with the ion exchanger.
The use of low protein concentrations to separate and recover whey proteins has disadvantages arising from the necessity to use large volumes of solution. These disadvantages include the need to use larger vessels, and long transfer times in and out of the vessel (either a batch reactor or a column) for the protein solution. Such processes may therefore be uneconomic, particularly when used to prepare whey protein components on an industrial scale.
Some references mention that an ultrafiltration retentate of whey or whey protein concentrate powder (WPC) can be used as a starting source of whey proteins, but give no examples (see eg EP 0,488,589 and GB 2,251,858). Others give examples, but either limit the concentration factor to 2.5 fold (Palmer, 1977, above) or 5-fold (GB 2,179,947) or alternatively concentrate much further but then dilute with water back to a protein concentration of 0.6% (Morinaga, JP 2,104,533) or 1% (EP 0,320,152). Thus, these references avoid high protein concentrations in the starting materials. For example, in US. Pat. No. 4,218,490 it is stated that “If desired, step (a) (the ion exchange step) can be preceded by an initial concentration step effected, for instance, by ultrafiltration”. However, the same author limits the utility of this pre-concentration to a level of 1.5% protein, that is, a 2.5 fold concentration (see Palmer, 1977, referred to above).
As noted above, the prior art generally teaches avoiding using high protein. concentrations in the raw materials. EP 0,320,152 does give an example (Example 2) where a WPC 60 (600% protein on total solids, which is probably a 10-15% protein solution) is passed through a column of anion exchanger. However, the utility of this is not mentioned or apparent and it is believed that, although the method worked in that the column bound whey proteins other than Ig, the protein capacity of the ion exchanger was low.
U.S Pat. No. 4,834,994, which is directed to the selective adsorption of &bgr;-lactoglobulin by a cation exchanger, refers in claim
3
to WPC as a possible starting material. It is clear however that this patent only contemplates the use of dilute whey protein solutions, with protein concentrations in the range of 0.5 to 1.5%. Example 4 in the patent uses only a 2-fold concentration of the whey and in a further option dilutes this with an equal volume of water. A very large quantity of water is required to do this, which is not practical, particularly on an industrial scale.
U.S Pat. No. 5,077,067 (Thibault) also relates to the selective adsorption of &bgr;-lactoglobulin but by an anion exchanger. Example 4 of Thibault takes a WPC-powder and reconstitutes it at 10% solids (8% protein). When this is mixed with ion exchanger (as in Example 4), there will be considerable amount of further dilution by water in the bed of the ion exchanger, which means that the actual protein concentration of the solution contacting the ion exchanger is only about 4%.
The applicants believe it would be desirable for reasons stated below to be able to use more concentrated whey protein solutions in methods of isolating whey proteins using ion exchange. They have also surprisingly found that this can be achieved, while maintaining efficient use of the ion exchanger, by contacting a concentrated whey protein solution having reduced ionic strength with the ion exchanger.
It is therefore an object of the present invention to provide a method of separating whey proteins which goes some way towards achieving the above desideratum, or at least to provide the public with a useful choice.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides a preparative method of isolating a preselected whey protein or group of whey proteins from a solution, wherein the method comprises the following steps:
(a) contacting a whey protein solution with a preselected ion exchanger for a time and at a temperature sufficient to enable the preselected whey protein to be adsorbed; wherein the whey protein solution has (1) a protein content in the range of about 5% to about 20% by weight, (2) a pH of a preselected level, which is the level at which the preselected whey protein or group of whey proteins selectively binds to the preselected ion exchanger, and (3) a reduced ionic strength; and
(b) recovering either or both of the following: (1) the whey protein component adsorbed in step (a), and (2) the breakthrough whey protein component not adsorbed in step (a).
In a further aspect the present invention provides a preparative method of isolating a preselected whey protein or group of whey proteins from a whey protein solution, wherein the method comprises the following steps:
(a) obtaining a whey protein solution which has a protein content in the range of about 5% to about 20% by weight;
(b) contacting the whey protein solution with a preselected ion exchanger to which the preselected whey protein or group of whey proteins selectively binds at a preselected pH, and wherein the ion exchanger also adjusts the pH of the whey solution to about the preselected pH and reduces the ionic strength of the whey solution; and
(c) recovering either or both of the following: (1) the whey protein component adsorbed in step (b); and (2) the breakthrough whey protein component not adsorbed in step (b).
In this specification the term “reduced ionic strength”, when used in relation to a whey protein solution, refers to a whey protein solution in which the concentration of salts (ash content) is reduced at the preselected pH at which the whey protein solution is to be contacted with the preselected ion exchanger, relative to a whey protein solution ultra filtered to the same protein concentration and
Ayers John Stephen
Elgar David Francis
Pritchard Mark
Knobbe Martens Olson & Bear LLP
Massey University
Saunders David
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