Casein Macropeptide (GMP):

The casein macropeptide is found in "sweet" whey, but not in acid whey. The casein macropeptide, frequently referred to as the glycomacropeptide (GMP),is derived from the action of chymosin on k-casein. It is the more hydrophilic C-terminal portion of the molecule, containing the oligosaccharides that are O-linked to threonine and serine. Its properties have been reviewed by Lopez and Ramos (1992), El-Salam, etal. (1996)and Dziuba and Minkiewicz (1996).

General Characteristics:

GMP is a heterogeneous material, consisting of several components with different concentrations and size - depending on the source and method of preparation. The amino acid of the GMP released from casein has been determined (Mercier, et.al., 1973). GMP is the C-terminal portion of k-casein from residue 106 (Met) to Val 169. The glycosylation is variable and is influenced by stage of lactation, genetic phenotype of k-casein ( Dziuba and Minkiewicz, 1996). The level of glycosylation is high in colostrum, is higher in AB and AA k-casein, and increases with mastitis and an increase in somatic cell counts.

GMP is lacking in aromatic amino acids and shows no absorption at 280 nm. It is also low in basic, acidic and hydroxy amino acids (Eigel, etal., 1984).

The carbohydrate content is complex with attachment of from 0 to 5 molecules linked to either threonine or serine. Four different oligosaccharides has been identified (Fiat, etal., 1988). The carbohydrates include sialic acid (N-acetylneuraminic acid, NANA) galactose and N-acetylgalactosamine.

All of the phosphate associated with k-casein has been associated with the casein macropeptide. Different components of GMP have different numbers of phosphate groups per molecule, ranging from 1 to 3.

Stan and Cherikov (1974) reported the GMP represented a major and minor component of 32 and 8 kDa with a sialic acid content of 9.75 and 1% respectively. GMP is reported to undergo aggregation interactions that are pH dependent. Kawakami, etal. 1992 showed that at pH 7.0 the apparent molecular weight ranged from 20 to 50 kDa, whereas at pH 3.5 it ranged from 10-30 kDa.

Biological and Physiological Properties:

The biological function of the k-casein glycomacropeptide can be attributed to both the carbohydrate and the peptide make up of the k-casein. The two most important carbohydrate components are N-acetylneuraminic acid (NANA) and N-acetlygalactosamine (Dziuba and Minkiewicz, 1996). NANA has been shown to have a large number of biological functions, which include: regulation of shell shape and lifetime, component of hormones, contribution to the binding of calcium, contribution to regulation of cell growth and differentiation, interaction with antibodies, component of cell receptors, protection against bacteria adhesion and contribution in interactions with viruses. The manner in which the carbohydrate moieties influence the biological function of GMP still requires further clarification, since specific peptides derived from trypsin and chymotrypsin action on GMP have varied biological function.

The biological and physiological properties that have been attributed to GMP or peptides derived from it have been reviewed by El Salam, et.al. (1996) and Dziuba and Minkiewicz (1996). These include:

-inhibition of adhesion of oral actinomyces and streptococci to erythrocytes( Neeser et al, 1988)

-reduction in gastric secretion (Chernikov, 1974; Aleinik, 1984; Stan, etal., 1988; Yvon, et.

al, 1994)

-inhibition of splenocyte proliferation (Yun, et al., 1996: Otani, et.al., 1992; Otani and Hata

(1995); Otani and Monnai, 1993; Otani, et.al., 1995)

-inhibition of proliferation of rabbit Peyer’s patch cells (Otani, et.al. 1995)

-inhibition of B-interferon production of human diploid fibroblasts (Yamada, etal., 1991)

-inhibition of binding of cholera toxin to its receptor (Kawasaki, et.al., 1992)

-dental plaque and dental caries inhibition (Neeser, 1987)

-inhibition of influenza virus haemagglutonin (Kawasaki, et.al., 1993)

-stimulation of cholecystokinin release from intestinal cells (Beucher, etal., 194a,b;

Yvon etal., 1994)

-growth promoting activity for Bifidobacteria (Azuma, et.al., 1984:Poch and

Bezkorovainy, 1991; Idota, etal. 1994)

-variable effects of the growth of lactic acid bacteria, depending on species and strain

(Ito, et.al, 1987, Bouhallab, et.al., 1993)

-product for control of phenylketonuria (PKU) (Kristinasen, 1976, Smithers, et al, 1991)

-formation of biologically active GMP derived peptides (Leonil and Molle, 1990;

Bouhallab, etal., 1992; Jolles, et.al., 1986; Otani, et.al. 1992)

-inhibition of platelet aggregation (Fiat et al, 1988: Fiat et al., 1989; Fiat, et al. 1993)

The biological activity of the GMP is thought to depend on the content and structure of the sugar moieties. Inhibition of splenocyte proliferation, toxin binding, inhibition of hemagglutination, stimulation of bifidobacteria have all been related to the NANA component of the GMP . mechanism is thought to be that the NANA interacts with specific receptors.

Most of the studies of the biological activity of GMP have been done invitro, and require invivo conformation. Is some cases, such as inhibition of bacterial adhesion (Neeser, et. al, 1988) or regulator activity in the digestive tract (Yvon, etal. 1994), the GMP can act without being adsorbed. Other actions, such as immunomodulation or interactions with blood components would be expected to require absorption. This would occur following the action of digestive enzymes with the formation of smaller fragments with or without associated carbohydrates. Intact macropeptides have been found in the plasma of new-borns fed cow’s milk-based formula (Chabance, etal. 1995)

Biological Properties of fragments formed by protease action:

The biological activities of casein peptides, including GMP has been reviewed by Schlimme and Meisel (1995). Some of the biological activities attributed to GMP may in fact be caused by the action of digestive enzymes on the GMP in the intestine, with the release of specific peptides. Dziuba and Minkiewicz (1996) review the cleave sites of different enzymes on the C-terminal part of bovine GMP.

GMP is readily hydrolysed at pH 6.6 by trypsin and chymotrypsin. Trypsin acts on lysine positions at 111, 112, and 116. Both small (4-7 residues) and large (>7 residues) are found and can be separated into two broad groups by tight UF membranes (Bouhallab, et.al., 1992).

Some specific functions of peptides derived from the GMP include:

.-antithrobotic activity of fragments; 108-110; 106-116, 106-112, 113-116 (Jolles, et. al, 1986; Leonil and Molle, 1990)

-antihypertensive activity of fragment 108-110 (Kohmura, etal., 1990; Meisel and

Schlimme, 1990)

-proliferation of splenocytes induced by lipolysaccharide inhibited by GMP region 106-109

(Otani and Monnai, 1993; Otani and Hata, 1995)

-binding of Cholera toxin to its receptor (Kawasaki, etal. 1992)

-acid secretion in the stomach ( Cherikov, 1974: Stan and Chernikov, 1979; Stan, et al.,

1988)

-activation of growth of lactic acid bacteria (Bouhallab, et. al, 1993)

The degree to which the carbohydrate moiety remains attached to the peptide fragments and its role in the biological function of these peptide fragments requires further investigation.

Small differences in the peptides can create large differences in activity. For example the 112-116 peptide from the trypic digest of GMP appears to be about 200 times more active that the 113-116 peptide. This has been attributed to the presence of a lysine in the 112-116 fragment (Maubois, et.al. 1991)

Nutritional Properties:

GMP is unique in its amino acid composition, lacking all of the aromatic amino acids and enriched in branched chain amino acids.

El Salam, et.al. (1996) indicated that GMP might be useful for diets to control various liver disease, where branched chain amino acids appear to be used as a carbon source.

Chernikov, et.al. (1974) observed the suppression of gastric secretion after injection of GMP, and hypothesised that GMP is a physiological agent that regulated the digestive function in mammals. The work in this laboratory continued for an additional 15 years, and found that the active components are peptides formed by pepsin hydrolysis at low pH. Stan, et.al. (1988) showed that pepsin hydrolysis produced several different peptides with different physiological activities. One of these was an oppioid effect and the other was a satiation effect when administered to starving animals. The latter effect was similar to that shown by cholecystokinin injection.

Kristiansen (1976) and Smithers, etal. (1991) suggested that GMP could be used as an ingredient in the development of diets for those suffering from phenylketonuria.

Marshall (1991) developed was a protein fortified fruit gel, that was targeted towards a product that was low in phenylalanine for people suffering from PKU. GMP-fortified apple gels (using carrageenan as a gelling agent) show the best appearance and gel firmness at pH 4.5. Lower pH levels produced products that were not as desirable.

Foods, based on whey proteins, developed for special dietary use and infant formulas have included whipped products, meringues, biscuits, fortified fruit jellies (Burton and Skudder, 1987; Marshall, 1991; Smithers, etal., 1991, El Salam, 1996).