Food or edible material: processes – compositions – and products – Products per se – or processes of preparing or treating... – Protein – amino acid – or yeast containing
Utility Patent
1998-07-31
2001-01-02
Weier, Anthony (Department: 1761)
Food or edible material: processes, compositions, and products
Products per se, or processes of preparing or treating...
Protein, amino acid, or yeast containing
C426S495000, C426S422000, C426S271000, C426S583000
Utility Patent
active
06168823
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a process for producing kappa-casein macropeptide having nutraceutical properties. The present invention specifically relates to a process for producing substantially-pure kappa-casein macropeptide from whey using anion exchange and immobilized metal ion affinity chromatography. The present invention is also directed to a method for providing a means for large-scale production of kappa-casein macropeptide in a substantially pure form using fewer steps than methods of similar capability in purity.
BIBLIOGRAPHY
Complete bibliographic citations of the references referred to herein can be found in the Bibliography section immediately preceding the claims.
BACKGROUND OF THE INVENTION
Nutraceuticals are foods that have specific medicinal and nutritional benefits. One nutraceutical, kappa-casein (&kgr;-casein), macropeptide comprises 15-20% of the protein in whey, making its supply plentiful and readily available for use in dietetic foods and nutraceuticals.
Widely differing extents of glycosylation of &kgr;-casein macropeptide (CMP) exist in whey and whey products, ranging from fully-glycosylated CMP (called &kgr;-casein glycomacropeptide, or GMP) to non-glycosylated CMP. For purposes of the present invention, CMP includes all forms of the &kgr;-casein macropeptide from the fully-glycosylated &kgr;-casein glycomacropeptide to the non-glycosylated &kgr;-casein glycomacropeptide. As discussed by Shammet et al. (1992), total CMP, which includes all degrees of glycosylation, is measured as non-protein nitrogen (NPN-2) soluble in 2% trichloroacetic acid (TCA). GMP is measured as non-protein nitrogen soluble in 12% TCA (NPN-12). The difference between NPN-2 and NPN-12 is non-glycosylated CMP.
CMP is the moiety cleaved from &kgr;-casein at the Phe
105
-Met
106
position by chymosin during cheese making (Dalgleish, 1982; Fox, 1989). CMP occurs at a concentration of 1.2 to 1.5 g/L in sweet whey, e.g. Cheddar cheese whey, (Marshall, 1991). Kawasaki et al. referred to previously reported nutritional advantages of GMP (U.S. Pat. No. 5,278,288 to Kawasaki and Dosako) and Tanimoto et al. (1992) suggested the utilization of GMP in dietetic foods and pharmaceuticals. The advantage of CMP as a substrate for hydrolysis is that it does not contain the amino acids tyrosine, phenylalanine, and tryptophan. These are hydrophobic aromatic amino acids responsible for the bitter taste of some peptides (Pedersen, 1994). Alternatively, Marshall (1991) suggested that CMP can be used as a protein source for the treatment of phenylketonuria, a hereditary disorder in which phenylalanine cannot be metabolized.
On a laboratory scale, CMP has been purified from &kgr;-casein in bovine whey using TCA precipitation (Shammet et al., 1992). GMP has been purified from bovine whey by alcohol precipitation after heat coagulation of whey protein (Saito et al., 1991). These methods are unlikely to be economical for large-scale manufacturing.
On a large scale, several methods have been developed using ultrafiltration (Kawakami et al., 1992; Kawasaki et al., 1993) and ion exchange (U.S. Pat. No. 5,278,288 to Kawasaki and Dosako; U.S. Pat. No. 5,290,107 to Kawasaki et al.) to purify GMP from whey or whey protein concentrate. However, the recovery of GMP using these methods is uneconomically low, at most 18%.
The basis of ultrafiltration purification is that the apparent molecular weight of GMP is 10 to 30 kDa at pH 3.5 and 20 to 50 kDa at pH 7.0. At pH 3.5, GMP permeates 20 to 50 kDa molecular weight cutoff (MWCO) ultrafiltration systems while larger proteins are retained by the membrane. Then at pH 6.5, GMP is retained by the same MWCO membranes while small molecular weight contaminants such as peptides pass through the filter.
Other methods have been developed to purify CMP from whey by anion exchange. U.S. Pat. No. 5,280,107 to Kawasaki et al. relates to contacting whey, adjusted to pH 4 or lower, with an anion exchanger to adsorb CMP. The adsorbed fraction is eluted and then concentrated and desalted to obtain the CMP. However at a pH of about 4 or lower, largely glycosylated CMP would be adsorbed to the anion exchanger, leaving unadsorbed and unrecovered a large amount of valuable non-glycosylated material. Kawasaki et al. state that because the sialic acids present in GMP have a pKa value of 2.7, these moieties have a net negative charge at pH values as low as 3 to 4. In non-glycosylated CMP, the acidic amino acid side chains (aspartic and glutamic acid) have a pKa of 3 to 5, and would have a substantial net negative charge only at a pH 5 and higher. The presence of sialic acids allowed Kawasaki et al. to separate GMP from proteins which lack sialic acids, by adsorbing the GMP to an anion exchanger at pH 4 or lower, because the other proteins are neutrally or positively charged at this pH. However, the method of Kawasaki et al. would not be suitable for recovering both glycosylated and non-glycosylated CMP from whey because non-glycosylated CMP would not bind strongly to an anion exchanger until it had a substantial net negative charge, which would occur only at pH 5 and higher.
Outinen et al (1995) adjusted whey to pH 5 and loaded it into an anion exchange column. The column was rinsed with water and the CMP fraction eluted with 2% sodium chloride. No downstream process was developed to remove contaminating peptides which contain aromatic amino acids. However, many applications for CMP require a low concentration of proteinaceous materials containing aromatic amino acids. For example, CMP can be used as a source or protein for patients with phenylketonuria as described previously.
Separation and purification accounts for a large proportion of the production cost of proteins. The main problem with processes based on precipitation methods is that achieving a pure product from a dilute solution such as whey is not commercially economical. The difficulty with ion exchange-based methods is the efficient production of a pure product. No prior art has used immobilized metal affinity chromatography to purify CMP.
SUMMARY OF THE INVENTION
The present invention is directed to a process for producing CMP from whey comprising contacting the whey with an anion exchanger to yield an adsorbed whey protein fraction enriched in CMP and a non-adsorbed whey protein fraction depleted in CMP; and then contacting the adsorbed whey protein fraction with an adsorbent to separate CMP from the whey protein fraction.
The present invention is also directed to a process for producing CMP from whey which comprises adjusting the whey to a pH greater than about 4; contacting the whey with an anion exchanger to yield a bound whey protein fraction enriched in &kgr;-casein macropeptide and an unbound whey protein fraction depleted in &kgr;-casein macropeptide; eluting the bound whey protein fraction enriched in &kgr;-casein macropeptide; and contacting the eluted bound whey protein fraction enriched in &kgr;-casein macropeptide with an immobilized metal affinity adsorbent to separate out the remaining whey protein fraction and obtain the substantially purified CMP.
The use of metal affinity chromatography to purify CMP from whey is novel and was discovered by experiment. When the CMP-rich whey protein fraction was passed through a metal affinity column, all the whey proteins except CMP were adsorbed, leaving the substantially pure CMP to pass through the column as effluent. The invention therefore provides for an “on-off” method for producing pure or substantially purified CMP.
The combination of anion exchange followed by IMAC adsorption is important for the success of the process for several reasons. IMAC adsorption is relatively expensive and because it does not bind the CMP by itself, it would not be able to remove lactose, fat and minerals from the CMP or be able to concentrate it compared to its concentration in whey. By preceding the IMAC step with an anion exchange step, the CMP is bound to and concentrated on the anion exchanger and rinsed free of lactose, fat and minerals, and the majority o
DeWitt Ross & Stevens S.C.
Weier Anthony
Wisconsin Alumni Research Foundation
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