A New Isolation Method of Caseinoglycopeptide from Sweet Cheese Whey

A New Isolation Method of Caseinoglycopeptide from Sweet Cheese Whey TADAO SAITO, ATSUO VAMAJI, and TAKATOSHI ITOH Laboratory of Animal Products Chemistry College of Agriculture Tohoku University Miyagi, Sendai 981, Japan

peanut (Arachis hypogoea) lectin, RP = reverse-phase.

ABSTRACT

Caseinoglycopeptides were isolated from sweet cheese whey by alcohol precipitation and ion-exchange chromatography after heat coagulation of whey protein. The most successful method for obtaining the highest yield was by heating 10% (wt/vol) whey solution at pH 6.0 for 1 h, followed by precipitation with cold ethyl alcohol (50% voVvol). The caseinoglycopeptide was fractionated into sialo- and asial
INTRODUCTION

Abbreviation key: CGP = caseinoglycopeptide, CH3CN = acetonitrile, K-CN = K-casein, CWP = cheese whey powder, Gal = D-galactose, GalNAc = N-acetylgalactosamine, a-LA = a-lactalbumin, p-LG = P-lactoglobulin, NANA = N-acetylneuraminic acid, PNA =

Received October 22, 1990. Accepted April 26, 1991. 1991 ] Dairy Sci 74:2831-2837

Whey is the aqueous portion of milk that is separated from the curd during cheese making or casein manufacture. Because whey is plentiful in lactose, protein, vitamins, and minerals, it is used in a wide variety of products, such as animal feeds, infant formulas, beverages, and confections. There are two types of whey, sweet and acid, and the former results from the action of chymosin in the production of ripened cheeses such as Cheddar, Swiss, and Gouda. Sweet whey contains a-lactalbumin (a-LA) and p-Iactoglobulin (P-LG), which comprise around 90% of the whey protein, proteose peptone, and, in particular, caseinoglycopeptide [(CGP, K-easein (K-CN), flO6-169l, which arises from K-CN by rennet treatment. K-Casein is the sole milk glycoprotein. It constitutes approximately 8 to 12% of whole casein, and all sugars in the molecule are located in the CGP part (residues 106 to 169) (6). Fournet et al. (8) have reported the sugar sequences of two acidic oligosaccharide (triand tetrasaccharide) chains from bovine K-CN. We have also reported the structures of the main sugar moiety (more than 84% of linked sugar chains) isolated from bovine whole casein (15). Recently, a number of new findings on bioactive peptides isolated from bovine milk protein have appeared, as discussed in a review by Meisel and Sclilimme (12). Opioid peptides derived from casein are called casomorphins (from a- and p-easeins, opioid agonists) or casoxins (from K-CN, opioid antagonists). aS1Caseino-phosphopeptide-9 derived from aand p-caseins is known as a mineral carrier. Also, CGP is thought to be a bioactive peptide, which may decrease appetite in dogs (19).

2831

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SAITO ET AL.

Preparation of CGP in the laboratory has usually been accomplished by chymosin (EC 3.4.23.4) digestion of pure lC-CN and removal of para-lC-protein with 12% TCA, followed by dialysis and lyophilization (11). It is also obtained industrially by pepsin or rennet digestion of sodium caseinate (3). Although sweet cheese whey is considered to be the most suitable CGP source, a technical and economical method has not yet been established because of the high contents of lactose, protein, and ash in whey. In this paper, we describe a new isolation method for sialo- and asialo-CGP from sweet cheese whey by combining affinity chromatography with chemical methods. We have focused especially on the view of industrial, large-scale production. MATERIALS AND METHODS Materials

Sweet cheese whey powder (CWP) from Holstein-Friesian cows was a commercial grade made by Yotsuba Milk. Products Co. Ltd. (Hokkaido, Jpn.). The DEAE-Toyopearl 650M was from Tosoh Corporation (Tokyo, Jpn.). Peanut (Arachis hypogoea) lectin (PNA)-Sepharose 4B (5.0 mg of PNA/ml of agarose gel) was obtained from Hohnen Oil Company Ltd. (Tokyo, Jpn.). The CGP as reference peptide for HPLC and amino acid analyses was prepared from purified lC-CN of normal bovine milk as described previously (16, 17, 18). The a-LA and ~-LG were purchased from Sigma (St. Louis, MO), and 180 of bovine blood serum was from UCB Bioproducts S.A. (Belgium). Analytical

Methods

Total CP and NPN soluble in 12% TCA were determined by the micro-Kjeldahl method (N factor = 6.38). Content of neutral sugar was measured by phenol-H2S04 reaction (5) and sialic acid [N-acetylneurarninic acid (NANA)] was by the method of Aminoff (1). Sugar composition of CGP was analyzed by GLC using the method of Qamp et al. (2). Both methanolysis and GLC were performed under conditions similar to those described earlier (16). loumal of Dairy Scieoce VoL 74. No.9. 1991

The CWP under different pH conditions and in 10% (wt/vol) solution in screw-cap medium bottles were heated and kept at 98'C for 1 h. Adjustment of pH of the CWP solution was performed with IN HCI or NaOH. Centrifugation was carried out at 9000 x g for 30 min to remove the denatured protein components. Ion-exchange chromatography of the peptide components in the supernatant was performed with a DEAE-Toyopearl 650M column (1.6 x 20 cm) using a linear concentration gradient of .02 to 1.0 M of ammonium bicarbonate buffer (pH 7.0). Effluent was monitored for peptide (210 om) and hexose (490 om). The CGP fraction was evaporated twice under reduced pressure at 4O·C to remove ammonium salts. The residue was dissolved in water, and the precipitate was removed by centrifugation. The CGP fraction, soluble in water, was only lyophilized. Amino acid analysis was performed using a Hitachi HPLC chromatograph (Model 638-30; Hitachi Ltd., Tokyo, Jpn.) equipped with a reaction unit for ninhydrin reagent. Anion-exchange column (4.0 x 150 mm. Hitachi custom resin 2619 F) was eluted by an automatically programmed stepwise elution system. The CGP was hydrolyzed in vacuum tubes with 6N HCI at 110'C for 20 and 72 h. Serine and threonine contents were calculated by extrapolating to zero time. Reverse-Phase HPLC

Analysis of CWP and CGP was carried out by reverse-phase (RP) HPLC on a Hitachi Model 638-30 chromatograph equipped with a Hitachi UV detector, a Hewlett-Packard integrator Model 3390 A (Hewlett-Packard, Avondale, PA), and an Asahipak ODP-50 column (C18 on polymer, 5-J.Ull particle size, 6.0 x 250 mm; Asahi Chemical Industry Co. Ltd., Kawasaki, Jpn.). The mobile phases were 10 and 60% acetonitrile (CH3CN) in water containing .05% trifluoroacetic acid. Analysis was conducted at a linear gradient of CH3CN from 10 to 60% developed over 30 min (.5 ml/ min) or 60 min (.3 ml/min) at 50·C. Column effluent was monitored at 210 om for detection of peptide. Affinity Chromatography

The whole CGP was f'mally fractionated by affinity chromatography with PNA-Sepharose

ISOLAnON OF CASEINOGLYCOPEPTIDE

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TABLE 1. The changes of total protein, NPN, and whey protein contents remaining in the supernatant from cheese whey powder (CWP) solution heated under three different pH conditions (Experiment I).

pH Items

5.0

7.0

9.0

- - - - (%) - - - -

Total protein NPN2 Whey protein)

11.73 3.90 7.83

6.91 3.90 3.01

8.33 3.54 4.79

9.91 3.91 6.00

110% (wt/vol) CWP solution, pH 5.8, unheated. 2nJe assumption caseinoglycopeptide.

3whey

here

is

that

NPN

contains

protein (%) = total protein (%) - NPN (%).

4B. The CGP (50 mg) in 10 mM phosphate buffer (pH 7.2) was applied on the PNA column (20 00) equilibrated with the same buffer. Sialyl-CGP was unadsorbed on the column. Adsorbed asialo-CGP was eluted from the column with the same buffer containing .2 M D-galactose (Gal) or lactose. Elution was monitored for peptide (210 nm) and sialic acid. The sialo- and asialo-CGP fractions were dialyzed against distilled water and then lyophilized. RESULTS AND DISCUSSION

Isolation 01 Caselnoglycopeptldes

As a preliminary experiment, 10% (wt/vol) CWP solution was heated (98°C) under three different pH conditions (pH 5.0, 7.0, and 9.0) for I h, followed by centrifugation. Table 1 shows total protein, NPN, and whey protein contents in the supernatants. Whey protein content was calculated by subtracting NPN from total protein content. The NPN content containing CGP was almost the same at the three pH conditions. The content of whey protein remaining in the supernatant was the lowest (3.01%) at pH 5.0. The suitable pH value for CGP isolation was, therefore, most likely in the acidic pH range, assuming that NPN was the sole source of COP. In a second experiment, 10% CWP solution was heated (9S°C) at seven different pH conditions (pH 1.0 to 7.0) for 1 h, followed by centrifugation. The COP content in the super-

Figure 1. Reverse-phase HPLC of sweet cheese whey (A) and caseinoglycopeptide standard (B) on Asahipak ODP-50. Twenty microliters of samples (.1%) were applied on the colnmn (6.0 x 250 mm). Dashed lines indicate the cooccntration of acetonitrile in the mobile phase.

natants was estimated by estimation of peak areas on the high performance liquid chromatogram. Figure 1 shows typical HPLC elution diagrams of .1 % solution of CWP (pH 5.8, unheated) and COP standard (see Materials and Methods). The CGP-linking sugar chain gave five peaks (peaks 1 to 5) on the chromatogram and was separately eluted from unglycosylated COP (peak 6), proteose-peptone (peaks 7 and 8), and other whey proteins: bovine serum albumin (peak 9), a.-LA (peak 10), and P-LG (peak 11). These multipeaks of the COP (peaks 1 to 5) were presumed to arise from the microheterogeneity of its sugar chains, especially the number of NANA residues (4, 23). Table 2 shows the change of the CGP contents in the seven supernatants after heating under different pH conditions. The values indicate the relative percentage of the COP in the proteins and peptides remaining in the supernatants. The highest proportion of the CGP content (39.60%) was obtained at pH 6.0, and the value was approximately three times higher than that of the original CWP (12.7%). Experiment 3 used 10% CWP solution, which was heated in a similar manner as in Experiment 2 but followed by addition of the same volume of cold ethyl alcohol. After standing for 3 h at 4°C, the precipitate was removed by centrifugation, and the CGP proportion of the remaining protein in the supernatant was again analyzed by HPLC. The proJournal of Dairy Science Vol. 74, No.9. 1991

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SAITO ET AL.

TABLE 2. The cbaDges of caseinoglycopeptide (COP) and N-acetylneuraminic acid (NANA) contents in N components remaining in the supernatant from cheese whey powder (CWP) heated under different pH conditions. Experiments

pH

1 (COP)l

2 (COP)

Control3 7.0 6.0 5.0 4.0 3.0 2.0 1.0

12.7 15.0 39.6 38.1 31.1 30.3 28.2 19.7

64.7 80.9 83.8 77.1 66.1 64.7 54.0 26.8

3 (NANA)2

(%)

5.5 52 5.8 4.0 1.6 0 0 0

lProportion of COP was estimaled by HPLC analysis. Values are relative percentage of COP of the remaining protein and peptides in supernatant. 2Content of NANA in COP was determined by the method of Amiooff (1).

Figure 2. Ion-excbaDge chromatography of Whey proteins and NPN remaining in the supernalant from cheese whey powder (CWP) solution heated at pH 6.0 on DEAl!-Toyopearl 650M. Ten percent (wt/vol) CWP solution was heated (9S'C) at pH 6.0 for 1 h, followed by cenuifugation (9000 x g, 30 min). Fifty milliliters of the supernalant were applied on the column (1.6 x 20 em). Linear gradient of .02 to 1.0 M ammonium bicarbonate (pH 7.0) (- - - - - -) , absorbance at 280 nm (0, protein); absorbance at 490 nm (e, hexose).

310% (wt/vol) of CWP solution, pH 5.8, unheated.

Finally, in Experiment 5, 10% CWP solucedure has been often used in our previous studies for removal of lactose and other con- tion was heated (98°C) at pH 6.0 for 1 h, taminants in preparation of milk oligosaccha- followed by centrifugation. After adjusting the rides from colostrum (21, 22). The change of pH of the supernatant to 9.0 with 1 M ~OH, CGP proportions in the seven samples after fractionation of the peptide and protein compoaddition of ethyl alcohol are also given in nents was carried out by ion-exchange chromaTable 2. The highest proportion of the CGP tography with DEAE-Toyopearl 650M. Figure (83.8%) was also obtained at pH 6.0, when the 2 shows the elution chromatogram with a linvalue was 2.1 times higher than that in Experi- ear gradient system. Lactose and the majority ment 2. The total yield was 6.6 times greater of unglycosylated CGP were not adsorbed on the column. The CGP adsorbed on the column than that of the original CWP (12.7%). In Experiment 4, 10% CWP solution was was eluted in two peaks having both hexose heated in a similar manner as in Experiment 2 and NANA. Residual whey proteins were but followed by addition of the same volume eluted from the column after the CGP peaks at a concentration of .35 M ammonium bicarbonof 24% TCA. After centrifugation, the superate. The most suitable concentration of amnatant was dialyzed against several changes of monium bicarbonate for the maximum recovdistilled water for 2 d and then lyophilized. ery of the CGP from whey protein was Sialic acid (NANA) contents of the CGP from considered to be .3 M. CWP heated under different pH conditions are The procedure shown in Figure 3 appears to shown in Table 2. The NANA content of the represent the optimal conditions from ExperiCGP was not changed by heating at higher ments 1 to 5 for isolation of CGP from CWP. than pH 6.0. At pH 5.0 and 4.0, 26.9 and The ion-exchange chromatography step was 70.8% of NANA were released, respectively. modified from analytical scale (column chroThe complete release of NANA occurred be- matography) to preparative scale (batch syslow pH 3.0. Therefore, it is concluded that the tem) by considering industrial, large-scale most suitable pH conditions for obtaining na- treatment. Unglycosylated CGP, which arises tive CGP and for changing whole CGP into from carbohydrate-free lC-CN and represents a asialo-CGP were pH 6.0 and pH 3.0, respec- contaminant in the batch adsorption of CGP to tively. DEAE-resin, became insoluble during the reJournal of Dairy Science Vol. 74, No.9, 1991

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ISOLAnON OF CASEINOGLYCOPEPIIDE

TABLE 3. Amino acid composition of caseinoglycopeptide (CGP) isolated from cheese whey powder (CWP) and reference CGP prepared from lC-casein (lC-CN). Residues, mol of CGP Amino acid

CGP from CWP

CGP from lC-CN

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Lysine

5.0 5.0 10.5 11.3 5.5 6.0 9.5 10.4 7.5 8.1 1.2.8 5.2 4.5 5.3 5.3 .7 1.5 5.5 5.8 1.5 1.0 2.7 3.1

CGp l from lC-CN A

CGP from lC-eN B

5 12 6 10 8 1 5 6 I 6

4 11 6 10 8 I 6 6 I 7 I 3

I

3

11bese data were calculated from the primaJy structure of Bos x-CN A-IP (bovine lC-casein variant A-I phosphate) and lC-CN B-IP (variant 8-1 phosphate) (6). Samples were hydrolyzed in 6N HCl at 1l0'C for 24 and 72 h in a sealed tube under vacuum. The values presented are the average of three determinations.

peated evaporation process, because of removal of ammonium salts. The precipitated unglycosylated CGP could thus be eliminated from the glycosylated CGP, which is soluble in water before lyophilization. The yield of CGP was approximately 1.1 g from 100 g of CWP. The amino acid composition between CGP by this procedure and reference CGP (see Materials and Methods) was determined. Results of amino acid analyses are in Table 3. From the amino acid composition, CGP by this method was identical to that of authentic CGP and also agreed with those values in the literature (6). Separation Of 51al0- and Aslalo-CGP by Afftnlty Chromatography

The CGP prepared from lC-CN usually contains a small amount of asialo-CGP, which has a disaccharide chain: Gal-P-I-3-N-acetylgalactosamine (GaINAc) (4). The sugar moiety of COP generally displays microheterogeneity, and the presence of neutral asialo-COP is not due to an artifact formed during the isolation process. The COP obtained by our optimal procedure (Figure 3) was fractionated by affinity chromatography with PNA. Figure 4 shows the elution pattern of COP from the PNA column. Elution was monitored by peptide and carb0hydrate analyses. Fraction 1, the unadsorbed

component, contained NANA, Gal, and GalNAc. Fraction 2 was the component eluted from the column with .2 M D-Gal and contained only Gal and GalNAc. These compo-

CWP (10 Wf/vol % solution) I Adjustment to pH (6.0) I Heating at 98'C for I h I Cooling to 4'C I

Addition of cold ethyl alcohol (50 vollvol %) I Standing at 4'C for 3 h I Centrifugation I

Adjustment to pH 9.0 I Ion-exchange chromatography (batch method) of the supernatant with DEAE-Toyopearl 650M I Elution with .3 M ammonium bicarbonate I Evaporation of the effluent to dIyness under vacuum I Removal of insoluble material by centrifugation I Lyophilization (whole CGP) Figure 3. A scheme of the optimal manufacturing procedure of whole caseinoglycopeptide (CGP) from sweet cheese whey powder (CWP). Journal of Dairy Science Vol. 74, No.9. 1991

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SAITO ET AL. .S

I

A

B

C .~

S I,

25

3J

3")

.J

.3

.2

,2

2S

30

3\

30

35

till)'

00

80

Elutlor

120

volume

160

(ml )

Figure 4. AffInity chromatography of the whole caseinoglycopeptide (COP) on peanut lectin (PNA) Sepharose 4B column. Whole COP was isolated from sweet cheese whey powder by the optimal procedure as shown in Figure 3. Fifty milligrams of COP in 10 mM phosphate buffer (10 m1, pH 7.2) was applied on the PNA column (20 ml). 'The arrow indicates the addition of .2 M D-galactose (Gal) in the same buffer. Absorbance at 210 nm (0, peptide); absorbance at 549 nm (e, sialic acid).

Figure 5. Reverse-phase HPLC of the fractionated components by the affinity chromatography on peanut lectin (PNA) column. A: Whole caseinoglycopeptide (COP) isolated by the procedure as shown in Figure 3. B: Fraction 2 (asialo-COP) from the PNA column as shown in Figure 4. C: Fraction 1 (sialo-COP) from the PNA column as shown in Figure 4. The HPLC conditions are given in Materials and Methods.

Preparation of Aslalo-Caselnoglycopeptlde

nents were analyzed by HPLC. Figure 5 shows typical HPLC chromatograms. The CGP (Figure 5A) was composed of five peaks, which were separated into the first peak. representing the asialo-CGP (Figure 5B, fraction 2 in Figure 4), and followed four peaks of sialoCGP (Figure 5C, fraction 1 in Figure 4). The complete separation of sialo- and asialo-CGP by conventional chromatographic technique was unsuccessful This was overcome by introduction of lectin-affinity chromatography. The PNA is known to agglutinate human erythr0cytes and lymphocytes treated with neuraminidase and to recognize an external I3-D-Gal residue of carbohydrate moiety, especially a disaccharide unit of Gal-I3-1-3-GalNAc, which is a common core unit of O-glycosidic sugar moiety in glycoconjugates (10, 13, 14). The asialo-CGP has the same carbohydrate unit. The ratio of asialo-CGP in the whole CGP fraction was estimated to be approximately less than 10% by HPLC analysis. The purity of both sialo- and asialo-CGP was over 90%, and neither whey protein nor lactose was detected by HPLC and GLC analyses. Journal of Dairy Science Vol. 74, No.9, 1991

Asialo-CGP could be artificially prepared from CWP solution heated at pH 3.0 and 98°C for 1 h, followed by the steps used for CGP isolation (Figure 3). Under these conditions, NANA was completely released from CGP (Table 2). The CGP obtained in this way gave only one peak on the HPLC chromatogram, as did that of the asialo-CGP shown in Figure 5B. The total yield of the asialo-CGP was approximately 1.0 g from 100 g of CWP. Recently, the ability to decrease the appetite of dogs has been suggested as a new bioactivity of CGP (19). Clarification of its mechanism of action may make it possible not only to elucidate the appetite control system but also to develop foods that prevent obesity in the future. In Japan, an infant formula supplemented with CGP prepared from casein by rennet digestion has been available since 1989; it is thought to be an anti-infection factor for bacteria Moreover, highly purified sialo- and asialo-CGP obtained in this study are also quite useful as enzymatic substrates for Nacetylneuraminidase (BC 3.2.1.18) and endoa-N-acetylgalactosaminidase (EC 3.2.1.97) (7, 9,20), respectively. Therefore, the demand for CGP in the field of dairy and food chemistry, biochemistry, and phannacology may well increase in the near future.

ISOLATION OF CASEINOGLYCOPEPTIDE ACKNOWLEDGMENTS

This work was supported in part by the CS (Chugai Seiyaku) Whey Research Association. REFERENCES

1 Aminoff, D. 1961. Methods for the quantitative estimation of N-acetyl-neuraminic acid and Iheir application to hydrolysates of sialomucoids. Biochem. J. 81: 384. 2 Clamp, J. R., G. Dawson, and L. Hough. 1967. The simultaneous estimation of 6-deoxy-lrgalactose (Lfucose), D-mannose, D-galactose, 2-acetamido-2deoxy-D-glucose (N-acetyl-D-glucosamine) and Nacetylneuraminic acid (sialic acid) in glycopeptides and glycoproteins. Biochim. Biophys. Acta 148:342. 3 Dohsako, S., T. Nishiya, and E. Deya. 1988. Process for !he production of lC-casein glycomacropeptide. Eur. Pat. Appl. EPO,291264,A2. 4 Doi, H., F. Ibuki, and M. Kanamori. 1979. Heterogeneity of reduced bovine lC-easein. J. Dairy Sci. 62: 195. 5 Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350. 6 Eigel, W. N., J. E. Butler, C. A. Emstrom, H. M. Farrell, Jr., V. R. Harwalkar, R. Jenness, and R. MeL. Whitney. 1984. Nomenclature of proteins of cow's milk: fifth revision. J. Dairy Sci. 67:1599. 7 Fan, J.-Q., S. Kadowaki, K. Yamamoto, H. Kumagai, and T. Tochikura. 1988. Purification and characterization of endo-a-N-acetylgalactosaminidase from Alcaligenes sp. Agric. BioI. Chern. 52:1715. 8 Fournet, B., A.-M. Fiat, C. Alais, and P. Ioll~. 1979. Cow lC-easein: structure of the carbohydrate portion. Biochim. Biophys. Acta 576:339. 9 Huang, C. C., and D. Aminoff. 1972. Enzymes that destroy blood groups specificity. (Y. The oligosaccharase of Clostridium perfringens.) I. BioI. Chem. 247:6737. 10 lrimura, T., T. Kawaguchi, T. Terao, and T. Osawa. 1975. Carbohydrate-binding specificity of !he socalled galactose-specific phytohemagglutinins. Carbohydro Res. 39:317. 11 Iolles, P., C. Alais, and I. IoI1~s. 1961. Etude compar6e des cas6ino-glycopeptides form6s par action de la presure sur les cas6ines de vache, de brebis et de

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ch~vre. I. Etude de la partie peptidique. Biochim. Biophys. Acta 51:309. 12 Meisel, H., and E. Schlimme. 1990. Milk proteins: precursors of bioactive peptides. Trends Food Sci. Technol. 1:41. 13 Novogrodsky, A., R. Lotan, A. Ravid, and N. Sharon. 1975. Peanut agglutinin, a new mitogen that binds to galactosyl sites exposed after neuraminidase treatment. I. Immunol. 115:1243. 14 Pereira, M.EA., and E. A. Kabat. 1976. Immunochemica1 studies on blood groups LXTI. Fractionation of hog and human A,H, and AH blood group active substance on insoluble immunoadsorbents of DoUchos and Lotus lectins. I. Exp. Med. 143:422. 15 Saito, T., T. Itoh, and S. Adachi. 1980. The chemical structure of !he main sugar moiety isolated from bovine whole casein. Agric. BioI. Chem. 44:1023. 16 Saito, T., T. Itoh, and S. Adachi. 1981. The chemical structure of a tetra-saccharide containing N-acetylglucosamine obtained from bovine colostrum. Biochim. Biophys. Acta 673:487. 17 Saito, T., T. Itoh, S. Adachi, T. Suzuki, and T. Usui. 1981. The chemical structure of neutral and acidic sugar chains obtained from bovine colostrum lC-casein. Biochim. Biopbys. Acta 678:257. 18 Saito, T., T. Itoh, S. Adachi, T. Suzuki, and T. Usui. 1982. A new hexa-saccharide chain isolated from bovine colostrum lC-easein taken at !he time of parturition. Biochim. Biopbys. Acta 719:309. 19 Stan, E. Y., and S. D. Groisman. 1983. Effect of 1(casein glycomacropeptide on gastrointestinal motility in dogs. Bull. Exp. BioI. Med. 96:889. 20 Umemoto, J., V. P. Bbavanandan, and E. A. Davidson. 1977. Purification and properties of an endo-a.-Nacetyl-D-galactosaminidase from Diplococcus pneumonjae. J. BioI. Chern. 252:8609. 21 Urashima, T., T. Saito, I. Nishimura, and H. Ariga. 1989. New galactosyl-lactose containing a-glycosidic linkage isolated from ovine (Booroola dorset) colostrum. Biochim. Biophys. Acta 992:375. 22 Urashima, T., T. Sakamoto, H. Ariga, and T. Saito. 1989. Structure determination of neutral oligosaccbarides obtained from horse colostrum. Carbohydr. Res. 194:280. 23 Vrecman, H. I., S. Visser, C. I. Slangen, and JAM. Van Riel. 1986. Characterization of bovine lC-casein fractions and !he kinetics of chymosin-induced macropeptide release from carbohydrate-free and carbohydrate-containing fractions determined by high-performance gel permeation chromatography. Biochem. I. 240:87.

Journal of Dairy Science Vol. 74, No.9, 1991