Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Glycoprotein – e.g. – mucins – proteoglycans – etc.
Reexamination Certificate
2000-07-24
2003-04-29
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Glycoprotein, e.g., mucins, proteoglycans, etc.
C530S322000, C530S360000, C530S361000, C530S412000, C530S414000, C530S416000, C530S418000, C530S832000, C530S833000
Reexamination Certificate
active
06555659
ABSTRACT:
TECHNICAL FIELD
This invention relates to a process for isolating glycomacropeptide (GMP) from a feedstock. More particularly, it relates to the isolation of GMP to a purity under which it has an amino acid composition containing less than 0.5% w/w of phenylalanine (Phe).
GMP is one of a number of names for the peptide split off from &kgr;-casein by the enzymes chymosin and/or pepsin. The peptide is also known as casein macropeptide (CM) or casein derived peptide (CDP).
GMP is found in sweet wheys. GMP carries all of the carbohydrate groups of the parent &kgr;-casein protein. &kgr;-casein is the only casein protein which is glycosylated. Another feature is that when the pH of a solution of GMP is less than 4 the molecular weight is 9000 Da. When the pH is greater than 4, the GMP apparent molecular weight increases to 45000 Da. When concentrating solutions of GMP by ultrafiltration it is preferable for the solution to have a pH>4 unless the membrane cut-off is ≦10,000 Da. A further feature of GMP is that it does not contain any aromatic amino acids including Phe in its structure.
GMP has a number of potential therapeutic uses as well as having functional properties which make it very useful as an ingredient in food compositions. One important utility is as a nutritional component for use in the diets of persons suffering from phenylketonuria (PKU) (Marshall S, (1991), Food Res Quarterly, 51, 86-91). Phenylketonurics lack Phe hydroxylase in their metabolic system. Therefore, they are unable to utilise Phe present in foods. This can result in a sufficient accumulation of Phe to cause irreversible mental retardation. In order for GMP to be safe for use in feeding to phenylketonurics the Phe level should be as low as possible. A representative product specification would require that the Phe level be 0.5% w/w or less and it would be desirable to have a method of isolating GMP to such a low level of Phe impurity on a production level scale.
BACKGROUND ART
Although a number of processes for isolating GMP are known, none of those processes have been shown to be capable of producing GMP having 0.5% w/w or less Phe impurity on a production level scale. One reason for this is that a GMP product of sufficient purity has not yet been manufactured. For example, assuming that the contaminant proteins contain an average 4% Phe, then a purity of greater than 88% GMP is required to give less than 0.5% Phe.
For example, EP A291264 discloses an industrial scale process for the purification of GMP from a mixture of &agr;, &bgr; and &kgr; caseins whereby the caseins are subjected to enzymatic milk-coagulating treatment to obtain non-coagulate components as an effluent. The effluent is then kept or rendered acidic to form a precipitate and the remaining effluent subjected to a desalting treatment to produce a GMP with 82% purity. No amino acid analysis data of the purified GMP is disclosed. This process is further described in Tanimoto et al, Biosci. Biotech. Biochem., 56(1), 140-141, 1992, which discloses a large-scale preparation of GMP from rennet casein whey. The whey was filtered and the filtrate desalinated and freeze dried to produce GMP powder having a Phe content of 2.4% w/w. The GMP powder was further purified by Q-Sepharose® ion-exchange chromatography to give a purified GMP with 0.9% w/w Phe.
GB 2188526 discloses a process for producing a proteinaceous material from milk or casein-containing milk products at pH 4-6 using anion exchange chromatography. No amino acid analysis data of the purified proteinaceous material is disclosed.
GB 2251858 discloses a process for producing GMP from milk raw materials by adjusting the pH of the material to ≦4, contacting the solution with an anion exchanger, concentrating and desalinating the eluate to give a GMP a 51% purity. This product may be further purified by ultrafiltration according to U.S. Pat. No. 5,075,424 (below) to produce GMP of 87% purity. No amino acid analysis data of the purified GMP is disclosed.
Outinen et al, Milchwissenschaft 50(10), 570-574, 1995, discloses a process for isolating GMP from cheese whey using an inexpensive polystyrenic strong basic anion exchange resin at pH 5. The Phe content of the purified GMP was 0.9% w/w. Further purification of this product by TCA precipitation gave a Phe content of 0.63-0.79% w/w.
U.S. Pat. No. 5,075,424 discloses a process for producing GMP from milk starting materials at pH≦4 by ultrafiltration. This process relies on the discovery that GMP has a smaller apparent molecular weight at pH<4 than at pH>4 so that only GMP will pass through a 10,000-50,000 molecular weight cut off membrane at pH<4. After ultrafiltration, the pH of the permeate is adjusted to pH≧4 and subjected to a second ultrafiltration using a membrane with a molecular weight cut-off of ≦50,000 Da to give a GMP retentate which is 82% pure. No amino acid analysis data of the purified GMP is disclosed.
Kawasaki et al, Milchwissenschaft 48(4), 191-195, 1993 discloses a purified GMP product using the method of U.S. Pat. No. 5,075,424 having a Phe content of 0.6% w/w and a purity of 81%.
Kawasaki et al, Milchwissenschaft 47(11), 688-693, 1992 discloses the same GMP product as above which is fractionated further by analytical chromatography systems (size exclusion chromatology and anion exchange chromatography). The purified GMP products had Phe content of 0.1-0.3%. However, such analytical systems are not suitable for mass production.
U.S. Pat. Nos. 4,042,575 and 4,042,576 each disclose a process of purifying glycoproteins (including GMP) by double ultrafiltration or flocculation of whey proteins followed by ultrafiltration. No amino acid analysis data for the purified glycoproteins is disclosed.
JP 04243898 discloses a process for producing GMP from cheese whey etc at pH 3-6 by heating, adding ethanol, centrifuging and loading the supernatant onto an anion exchange column and eluting GMP with 0.3M ammonium bicarbonate. This process is also reported in J. Dairy Sci 74, 2831-2837, 1991, where amino acid analysis data is given for the isolated GMP. However, Phe is not included in their analysis.
AU 74081/91 discloses a process for producing GMP from a whey protein concentrate in which the proteins are flocculated, the supernatant concentrated by ultrafiltration and the retentate treated with ethanol to produce a precipitate and a second supernatant. The second supernatant is collected and dried to give GMP powder of 84% purity. No amino acid data is disclosed.
JP 3-294299 discloses a process for the manufacture of GMP from whey by heating a 5-50 wt% solution of whey proteins followed by freezing and thawing. The supernatant is then separated, desalted and concentrated by ultrafiltration. No purity or amino acid analysis data of the recovered GMP is given.
WO 94/159252 discloses a method of producing GMP from whey using ultrafiltration, heat treatment of the retentate at 95° C. for 15 minutes, adjustment of the pH to 4-5 filtering and collecting GMP from the filtrate. The GMP is 70% pure and the Phe content is ⅓ that of the raw material. The exact concentration of Phe is not disclosed.
EP 0488589 discloses a process for producing GMP by contracting milk raw materials with an ion exchanger, collecting the protein which does not adsorb on the ion exchanger, concentrating and desalting to obtain GMP of 55%-88% purity. No amino acid analysis data is provided.
All of the aforementioned processes are aimed at producing enriched GMP fractions and are not necessarily concerned with reducing Phe content of the GMP as they are not concerned with producing diets for phenylketonurics.
According to WO 93/17587, Phe may be removed from proteinaceous material such as whey protein by enzyme hydrolysis and ultrafiltration to remove unhydrolysed protein. The permeate containing mainly amino acids and small peptides is passed through a column of adsorption resin to remove Phe at pH 6-7 (to give a product with 0.3% Phe) or pH 3-5 (to give a product with ≦0.1% Phe). Methods involving enzymatic hydrolysis of proteins
Ayers John Stephen
Coolbear Kay Patricia
Elgar David Francis
Pritchard Mark
Knobbe Martens Olson & Bear LLP
Low Christopher S. F.
Mohamed Abdel A.
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