Routine o-glycan characterization in nutritional supplements--a comparison of analytical methods for the monitoring of the bovine kappa-casein macropeptide glycosylation.

Analytical procedures, including capillary isoelectric focusing (CIEF), high-performance anion-exchange chromatography coupled to amperometric detection (HPAEC-PAD) and normal-phase chromatography with fluorescence detection are presented for the characterization of a highly O-glycosylated caseinomacropeptide (CGMP) and the detection of subtle glycosylation differences between CGMP Batches obtained with two different preparation procedures. Modified two-step CIEF allowed monitoring of glycopeptide heterogeneity and determination of the isoelectric points of acidic glycoforms. The mixture of wide and narrow pH range ampholytes was optimized to improve glycoform resolution. The pI of the different CGMP glycoforms was evaluated with pI internal standards and found to range between 3.08 and 3.58, which indicates a very acidic glycopeptide. Moreover, the monosaccharide composition was determined with HPAEC-PAD after neutral and amino sugars release by using adequate acidic hydrolysis of CGMP. Results indicated a similar composition for Batches I and II, but the monosaccharide percentages were 3-4 fold higher in Batch I, particularly for galactose and glucose. This likely reflects a higher content in lactose in the case of Batch I. Finally, O-linked oligosaccharides were released with an automated hydrazinolysis and derivatized with a sensitive labelling reagent, 2-aminobenzamide. The derivatives were then analyzed by normal-phase HPLC coupled with fluorescence detection, and separated on the basis of hydrophilic interaction, which allowed oligosaccharide mapping of the two CGMP. It appeared that the two CGMP preparations had an almost identical O-glycan population, but CGMP Batch I was more glycosylated than Batch II. Additionally, the sizes of the separated glycans, expressed as the number of glucose units, were tentatively assigned using calibration with a partial hydrolysate of dextran. In conclusion, a combination of electrophoretic and chromatographic techniques was found powerful in studying glycoprotein heterogeneity and assessing batch-to-batch consistency.

Structure and properties of the exopolysaccharide produced by Streptococcus macedonicus Sc136.

Streptococcus macedonicus is a Gram positive lactic acid bacterium that is part of the starter flora present in Greek sheep and goat cheeses. The S. macedonicus Sc136 strain produces a high-molecular-mass, highly texturizing exopolysaccharide composed of D-glucose, D-galactose, and N-acetyl-D-glucosamine in the molar ratio of 3:2:1. The structure of the exopolysaccharide produced by S. macedonicus Sc136 was determined by chemical analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy. The repeating unit was shown to be: (see text) The polysaccharide sidechain beta-D-Galf-(1-->6)-beta-D-Glcp-(1-->6)-beta-D-GlcpNAc is a key factor in the highly texturizing properties of the S.macedonicus Sc136 exopolysaccharide. Finally, the trisaccharide sequence beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp corresponds to the internal backbone of the lacto-N-tetraose and lacto-N-neotetraose units, which serve as a structural basis for the large majority of human milk oligosaccharides, an additional property offering an important potential for the development of improved infant nutrition products.

Glycosphingolipid binding specificities of rotavirus: identification of a sialic acid-binding epitope.

The glycosphingolipid binding specificities of neuraminidase-sensitive (simian SA11 and bovine NCDV) and neuraminidase-insensitive (bovine UK) rotavirus strains were investigated using the thin-layer chromatogram binding assay. Both triple-layered and double-layered viral particles of SA11, NCDV, and UK bound to nonacid glycosphingolipids, including gangliotetraosylceramide (GA1; also called asialo-GM1) and gangliotriaosylceramide (GA2; also called asialo-GM2). Binding to gangliosides was observed with triple-layered particles but not with double-layered particles. The neuraminidase-sensitive and neuraminidase-insensitive rotavirus strains showed distinct ganglioside binding specificities. All three strains bound to sialylneolactotetraosylceramide and GM2 and GD1a gangliosides. However, NeuAc-GM3 and the GM1 ganglioside were recognized by rotavirus strain UK but not by strains SA11 and NCDV. Conversely, NeuGc-GM3 was bound by rotaviruses SA11 and NCDV but not by rotavirus UK. Thus, neuraminidase-sensitive strains bind to external sialic acid residues in gangliosides, while neuraminidase-insensitive strains recognize gangliosides with internal sialic acids, which are resistant to neuraminidase treatment. By testing a panel of gangliosides with triple-layered particles of SA11 and NCDV, the terminal sequence sialyl-galactose (NeuGc/NeuAcalpha3-Galbeta) was identified as the minimal structural element required for the binding of these strains. The binding of triple-layered particles of SA11 and NCDV to NeuGc-GM3, but not to NeuAc-GM3, suggested that the sequence NeuGcalpha3Galbeta is preferred to NeuAcalpha3Galbeta. Further dissection of this binding epitope showed that the carboxyl group and glycerol side chain of sialic acid played an important role in the binding of such triple-layered particles.

Lactobacillus johnsonii La1 shares carbohydrate-binding specificities with several enteropathogenic bacteria.

The carbohydrate-binding specificities of the probiotic lactic acid bacterium Lactobacillus johnsonii La1 (a health-beneficial bacterial strain able to be incorporated into the human intestinal microflora) were investigated in vitro. First various soluble complex carbohydrates were tested as potential inhibitors of the strain adhesion onto Caco-2 intestinal epithelial cells, and then bacterial binding to glycolipids immobilized on TLC plates was probed. Two major carbohydrate-binding specificities of Lactobacillus johnsonii La1 were identified. A first one for an Endo-H treated yeast cell wall mannoprotein carrying mainly O:-linked oligomannosides, and a second one for the gangliotri- and gangliotetra-osylceramides (asialo-GM1). Similar carbohydrate-binding specificities are known to be expressed on cell surface adhesins of several enteropathogens, enabling them to adhere to the host gut mucosa. These findings corroborate the hypothesis that selected probiotic bacterial strains could be able to compete with enteropathogens for the same carbohydrate receptors in the gut.

Bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity.

BACKGROUND AND AIMS: The gastrointestinal microflora exerts a barrier effect against enteropathogens. The aim of this study was to examine if bifidobacteria, a major species of the human colonic microflora, participates in the barrier effect by developing antimicrobial activity against enterovirulent bacteria. METHODS: Antibacterial activity was examined in vitro against a wide range of Gram negative and Gram positive pathogens. Inhibition of Salmonella typhimurium SL1334 cell association and cell invasion was investigated in vitro using Caco-2 cells. Colonisation of the gastrointestinal tract in vivo by bifidobacteria was examined in axenic C3/He/Oujco mice. Antimicrobial activity was examined in vivo in axenic C3/He/Oujco mice infected by the lethal S typhimurium C5 strain. RESULTS: Fourteen human bifidobacterium strains isolated from infant stools were examined for antimicrobial activity. Two strains (CA1 and F9) expressed antagonistic activity against pathogens in vitro, inhibited cell entry, and killed intracellular S typhimurium SL1344 in Caco-2 cells. An antibacterial component(s) produced by CA1 and F9 was found to be a lipophilic molecule(s) with a molecular weight of less than 3500. In the axenic C3/He/Oujco mice, CA1 and F9 strains colonised the intestinal tract and protected mice against S typhimurium C5 lethal infection. CONCLUSION: Several bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity, suggesting that they could participate in the "barrier effect" produced by the indigenous microflora.

The development of functional foods: lessons from the gut.

Functional foods have resulted from the gradual recognition that healthy diets result from eating nutritious foods and from the identification of the mechanisms by which foods modulate metabolism and health. After initial successes with foods that reduce blood cholesterol level, probiotic bacteria and prebiotic carbohydrates have now also demonstrated added health benefits. As ingredients become more complex, the need to stabilize such ingredients in foods become increasingly important to the success of functional foods. Modern biotechnologies such as genomics, genetic expression and biomarkers of health and performance will be applied to this increasingly visible portion of human diets.

Unraveling the function of glycosyltransferases in Streptococcus thermophilus Sfi6.

Streptococcus thermophilus Sfi6 produces a texturizing exopolysaccharide (EPS) consisting of a -->3)[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->3)-alpha-D-GalpNAc-(1--> 3)-beta-D-Galp-(1--> repeating unit. We previously identified and analyzed a 14.5-kb gene cluster from S. thermophilus Sfi6 consisting of 13 genes responsible for its EPS production. Within this gene cluster, we found a central region of genes (epsE, epsF, epsG, and epsI) that showed similarity to glycosyltransferases. In this study, we investigated the sugar specificity of these enzymes. EpsE catalyzes the first step in the biosynthesis of the EPS repeating unit. It exhibits phosphogalactosyltransferase activity and transfers galactose onto the lipophilic carrier. The second step is fulfilled by EpsG, which transfers an alpha-N-acetylgalactosamine onto the first beta-galactoside. The activity of EpsF was determined by characterizing the EPS produced by an S. thermophilus epsF deletion mutant. This EPS consisted of the monosaccharides Gal, Glc, and GalNAc in an approximately equimolar ratio, thus suggesting that epsF codes for the branching galactosyltransferase. epsI probably codes for the beta-1,3-glucosyltransferase, since it is the only glycosyltransferase to which no gene has been assigned and it exhibits similarity to other beta-glycosyltransferases. EpsE shows the conserved features of phosphoglycosyltransferases, whereas EpsF and EpsG exhibit the primary structure of alpha-glycosyltransferases, belonging to glycosyltransferase family 4, whose members are conserved in all major phylogenetic lineages, including the Archaea and Eukaryota.

Powdered milk micellar casein prevents oral colonization by Streptococcus sobrinus and dental caries in rats: a basis for the caries-protective effect of dairy products.

Three animal studies were performed to investigate the influence of the macromolecular structure of milk casein on caries incidence and the possible ecological changes of the oral microbiota by such casein fractions. Towards this end, rats were infected with mixed bacterial suspensions of Streptococcus sobrinus OMZ 176 and Actinomyces viscosus Ny1. Various milk protein fractions were incorporated into carefully balanced powdered cariogenic diets to constitute the sole major protein component. Diets containing micellar casein had a pronounced and highly significant effect on almost all clinical and microbiological parameters examined. Both the formation of advanced dentinal fissure (B) and smooth surface (E) caries lesions was inhibited by diets containing micellar casein; this caries-inhibiting effect appeared to be due mainly to modifications within the plaque microbiota. The proportion of S. sobrinus in the oral cavity of rats was reduced (73-80%) by micellar casein-containing preparations, whereas the A. viscosus population was increased. Both these microbiological parameters were always negatively correlated. This appears to be the first example of a food component other than dietary sugars, selectively modifying the composition of the dental plaque microbiota of rats in such a way as to reduce its pathogenic potential. It also demonstrates the importance of establishing a molecular basis for the role of food components, which prove to be beneficial to oral health.

Introduction of the exopolysaccharide gene cluster from Streptococcus thermophilus Sfi6 into Lactococcus lactis MG1363: production and characterization of an altered polysaccharide.

Streptococcus thermophilus Sfi6 produces an exopolysaccharide (EPS) composed of glucose, galactose and N-acetylgalactosamine in the molar ratio of 1:2:1. The genes responsible for the EPS biosynthesis have been isolated previously and found to be clustered in a 14.5 kb region encoding 13 genes. Transfer of this gene cluster into a non-EPS-producing heterologous host, Lactococcus lactis MG1363, yielded an EPS with a similar high molecular weight, but a different structure from the EPS from the native host. The structure of the recombinant EPS was determined by means of 1H homonuclear and 1H-13C heteronuclear two-dimensional nuclear magnetic resonance (NMR) spectra and was found to be --> 3)-beta-D-Glcp-(1 --> 3)-alpha-D-Galp-(1 --> 3)-beta-D-Galp-(1 --> as opposed to --> 3)[alpha-D-Galp-(1 --> 6)]-beta-D-Glcp-(1 --> 3)-alpha-D-GalpNAc-(1 --> 3)-beta-D-Galp-(1 --> for the wild-type S. thermophilus Sfi6. Furthermore, L. lactis MG1363 (pFS101) was also lacking a UDP-N-acetylglucosamine C4-epimerase activity, which would provide UDP-GalNAc for a GalNAc incorporation into the EPS and probably caused the substitution of GalNAc by Gal in the recombinant EPS. This modification implies that (i) bacterial glycosyltransferases could potentially have multiple specificities for the donor and the acceptor sugar molecule; and (ii) the repeating unit polymerase can recognize and polymerize a repeating unit that differs in the backbone as well as in the side-chain from its native substrate.

Molecular organisation of the ice nucleation protein InaV from Pseudomonas syringae.

A new ice nucleation gene from Pseudomonas syringae was isolated and overexpressed as a fully active protein in Escherichia coli in order to gain experimental data about the structure of ice nucleation proteins. No evidence of a signal sequence or secondary glycosylation was found. Differences in the extent of aggregation were shown to modulate the ice nucleation activity. The circular dichroism spectrum of the purified protein indicated the presence of beta-sheet structure. This finding supports a recently proposed hypothetical model for the structure of ice nucleation proteins, which provides a plausible explanation for their aggregation tendency.

Structural characterization of the exocellular polysaccharides produced by Streptococcus thermophilus SFi39 and SFi12.

We investigated the structures of the exopolysaccharides (EPSs) produced by Streptococcus thermophilus SFi39 and SFi12. Both polymers were found to have molecular masses of greater than 2 x 10(6) Da. The SFi39 EPS consisted of D-glucose and D-galactose in a molar ratio of 1:1, whereas the SFi12 EPS was composed of D-galactose, L-rhamnose, and D-glucose in a molar ratio of 3:2:1. Methylation analysis of and nuclear magnetic resonance spectra recorded from the native polysaccharide, as well as oligosaccharides released by partial acid hydrolysis, allowed the complete structural determination of the SFi39 EPS, which consists of the following tetrasaccharide repeating unit: [formula: see text] Similar spectra recorded only from the native polysaccharide were sufficient to allow the structural determination of the SFi12 EPS, which consists of the following hexasaccharide repeating unit: [formula: see text] This study shows that the texturizing properties of different S. thermophilus ropy strains are based on the production of EPSs exhibiting chemical similarities but structural differences.

Lactobacillus helveticus Lh59 secretes an exopolysaccharide that is identical to the one produced by Lactobacillus helveticus TN-4, a presumed spontaneous mutant of Lactobacillus helveticus TY1--2.

Lactobacillus helveticus Lh59 produces a high-molecular-mass exopolysaccharide (> or = 2 x 10(6) Da) when cultured in skimmed milk. Compositional analysis, methylation analysis and NMR experiments (1H and 13C) recorded from the native polysaccharide as well as from oligosaccharides released by partial acid hydrolysis, allowed the complete structural determination of this polysaccharide, which consists of the following hexasaccharide repeating unit: [symbol: see text] This structure is identical to the one of an EPS produced by L. helveticus TN-4, which was claimed to be a spontaneous mutant of strain TY1-2.

The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo.

The adhering human Lactobacillus acidophilus strain LA1 inhibits the cell association and cell invasion of enteropathogens in cultured human intestinal Caco-2 cells (M. F. Bernet, D. Brassard, J. R. Neeser, and A. L. Servin, Gut 35:483-489, 1994). Here, we demonstrate that strain LA1 developed its antibacterial activity in conventional or germ-free mouse models orally infected by Salmonella typhimurium. We present evidence that the spent culture supernatant of strain LA1 (LA1-SCS) contained antibacterial components active against S. typhimurium infecting the cultured human intestinal Caco-2 cells. The LA1-SCS antibacterial activity was observed in vitro against a wide range of gram-negative and gram-positive pathogens, such as Staphylococcus aureus, Listeria monocytogenes, S. typhimurium, Shigella flexneri, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter cloacae. By contrast, no activity was observed against species of the normal gut flora, such as lactobacilli and bifidobacteria. The LA1-SCS antibacterial activity was insensitive to proteases and independent of lactic acid production.

Development of a capillary zone electrophoresis method for caseinoglycomacropeptide determination.

Caseinoglycomacropeptide (CGMP) is a polypeptide of 64 amino acid residues, derived from the C-terminal part of bovine kappa-casein. A sensitive and selective capillary zone electrophoresis method has been developed and validated for the analysis and quantitation of CGMP. Separation is carried out at 30 kV, using an uncoated fused-silica capillary and 20 mM sodium citrate buffer at acidic pH 3.5. The described method allows the separation of various CGMP subcomponents. The validation data proves that the method has the requisite selectivity, sensitivity, reproducibility and linearity for CGMP assay and for quality control during CGMP manufacturing (batch-to-batch reproducibility).

Incorporation of caseinoglycomacropeptide and caseinophosphopeptide into the salivary pellicle inhibits adherence of mutans streptococci.

The protective effects of milk and milk products against dental caries have been demonstrated in many animal studies. We have shown that this effect was mediated by micellar casein or caseinopeptide derivatives. A reduction in the Streptococcus sobrinus population in the oral microbiota of animals fed diets supplemented with these milk components was consistently observed. A possible explanation for these findings is that milk components are incorporated into the salivary pellicle, thereby reducing the adherence of S. sobrinus. This hypothesis was tested in vitro by the incubation of bovine enamel discs with unstimulated saliva. The resulting pellicle was washed and incubated with caseinoglycomacropeptide (CGMP) and/or caseinophosphopeptide (CPP) labeled with 17- and 12-nm gold particles. All samples were prepared for electron microscopy by high-pressure freezing followed by freeze-substitution. It was demonstrated by high-resolution scanning electron microscopy with back-scattered electron imaging, as well as by transmission electron microscopy, that both peptides were incorporated into the pellicle in exchange for albumin, confirming previous findings. This protein was identified with a mouse anti-human serum albumin followed by goat anti-mouse IgG labeled with 25-nm gold particles. Incorporation of CGMP and/or CPP into salivary pellicles reduced the adherence of both S. sobrinus and S. mutans significantly. It is suggested that the calcium and phosphate-rich micellar casein or caseinopeptides are incorporated into the pellicle. The resulting ecological shifts, together with the increased remineralization potential of this biofilm, may explain its modified cariogenic potential.

Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6.

We report the identification and characterization of the eps gene cluster of Streptococcus thermophilus Sfi6 required for exopolysaccharide (EPS) synthesis. This report is the first genetic work concerning EPS production in a food microorganism. The EPS secreted by this strain consists of the following tetrasaccharide repeating unit:-->3)-beta-D-Galp-(1-->3)-[alpha-D-Galp-(1-->6)]-beta-D- D-Galp-(1-->3)-alpha-D-Galp-D-GalpNAc-(1-->. The genetic locus The genetic locus was identified by Tn916 mutagenesis in combination with a plate assay to identify Eps mutants. Sequence analysis of the gene region, which was obtained from subclones of a genomic library of Sfi6, revealed a 15.25-kb region encoding 15 open reading frames. EPS expression in the non-EPS-producing heterologous host, Lactococcus lactis MG1363, showed that within the 15.25-kb region, a region with a size of 14.52 kb encoding the 13 genes epsA to epsM was capable of directing EPS synthesis and secretion in this host. Homology searches of the predicted proteins in the Swiss-Prot database revealed high homology (40 to 68% identity) for epsA, B, C, D, and E and the genes involved in capsule synthesis in Streptococcus pneumoniae and Streptococcus agalactiae. Moderate to low homology (37 to 18% identity) was detected for epsB, D, F, and H and the genes involved in capsule synthesis in Staphylococcus aureus for epsC, D, and E and the genes involved in exopolysaccharide I (EPSI) synthesis in Rhizobium meliloti for epsC to epsJ and the genes involved in lipopolysaccharide synthesis in members of the Enterobacteriaceae, and finally for eps K and lipB of Neisseria meningitidis. Genes (epsJ, epsL, and epsM) for which the predicted proteins showed little or no homology with proteins in the Swiss-Prot database were shown to be involved in EPS synthesis by single-crossover gene disruption experiments.

Binding of the fibrillar CS3 adhesin of enterotoxigenic Escherichia coli to rabbit intestinal glycoproteins is competitively prevented by GalNAc beta 1-4Gal-containing glycoconjugates.

We have attempted to characterize the binding specificity of the coli surface 3 (CS3) subcomponent of colonization factor antigen II of enterotoxigenic Escherichia coli, by means of an immunoblot method in which the binding of fimbriated bacteria to sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated rabbit intestinal cell membranes was evaluated. Isolated CS3 fibrillae as well as bacteria expressing CS3 on their surface bound to several intestinal cell membrane structures, i.e., structures present in the electrophoretic front and in the 30- to 35-kDa range and, most prominently, 120- to 140-kDa structures. Delipidization and protein digestion of the rabbit brush borders revealed that CS3 bound to structures of a proteinaceous nature. Sodium meta-periodate oxidation of the intestinal cell membranes abolished all their CS3 binding activity, indicating that CS3 bound to carbohydrate moieties of glycoproteins. The binding of CS3 to the separated intestinal proteins could also be inhibited by preincubation with the lectin derived from Maackia amurensis, indicating that CS3 bound to galactoproteins in the rabbit intestine. Inhibition experiments using equimolar amounts of various gangliosides demonstrated that GM1, asialo-GM1, and GM2 inhibited the binding of CS3 equally well, whereas GM3 was not as effective. These results suggested that the critical CS3 binding epitope consisted of the carbohydrate sequence GalNAc beta 1-4Gal. This was supported by electron microscopic experiments showing that this disaccharide, O linked to bovine serum albumin via a spacer, localized around CS3-positive bacteria but not at all around corresponding CS3-negative mutants. Furthermore, CS3-expressing bacteria recognized this neoglycoprotein when it was immobilized on nitrocellulose. The GalNAc beta 1-4Gal disaccharide has also been implicated as a binding structure for other pathogenic bacteria such as enteropathogenic E. coli and Pseudomonas aeruginosa.

A 23 kDa membrane glycoprotein bearing NeuNAc alpha 2-3Gal beta 1-3GalNAc O-linked carbohydrate chains acts as a receptor for Streptococcus sanguis OMZ 9 on human buccal epithelial cells.

Streptococcus sanguis colonizes several human oral surfaces, including both hard and soft tissues. Large salivary mucin-like glycoproteins bearing sialic acid residues are known to bind various S.sanguis strains. However, the molecular basis for the adhesion of S.sanguis to human buccal epithelial cells (HBEC) has not been established. The present study shows that S.sanguis OMZ 9 binds to exfoliated HBEC in a sialic acid-sensitive manner. The desialylation of such cells invariably abolishes adhesion of S.sanguis OMZ 9 to the cell surface. A soluble glycopeptide bearing short sialylated O-linked carbohydrate chains behaves as a potent inhibitor of the attachment of S.sanguis OMZ 9 to exfoliated HBEC. The resialylation of desialylated HBEC with CMP-sialic acid and Gal beta 1,3GalNAc alpha 2,3-sialyltransferase specific for O-glycans restores the receptor function for S.sanguis OMZ 9, whereas a similar cell resialylation with the Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase specific for N-glycans is without effect. Finally, the same resialylation reaction carried out with CMP-9-fluoresceinyl-sialic acid as a substrate yields exfoliated HBEC bearing fluorescence on a single 23 kDa protein, when using the alpha 2,3-sialyltransferase as the catalyst. The latter finding demonstrates that this 23 kDa cell surface glycoprotein bears NeuNAc alpha 2-3Gal beta 1-3GalNAc O-linked sugar chains, a carbohydrate sequence which is recognized by S.sanguis OMZ 9 on exfoliated HBEC. In similar experiments carried out with a buccal carcinoma cell line termed SqCC/Y1, S.sanguis OMZ 9 did not attach in great numbers to such cultured cells, and these cells were shown to not express membrane glycoprotein bearing alpha 2,3-sialylated O-linked carbohydrate chains.

In vitro modulation of oral bacterial adhesion to saliva-coated hydroxyapatite beads by milk casein derivatives.

Bovine caseinate, derivatives of its glycosylated moiety [caseinoglycomacropeptide (CGP)], and caseinophosphopeptides were evaluated as inhibitors of adhesion of oral bacteria to saliva-coated hydroxyapatite beads (S-HA). All milk casein-derived components behaved as potent inhibitors of Streptococcus sanguis OMZ 9 and Streptococcus sobrinus OMZ 176 adhesion to S-HA, whereas neither bovine serum albumin nor polyethyleneglycol were able to interfere with the adhesion of these strains. By contrast, none of the molecular species tested was able to inhibit the attachment of Actinomyces viscosus Ny 1 to S-HA. On the other hand, casein derivatives were shown to displace human serum albumin from S-HA beads. They were also able to bind to the bacterial cell surface of all strains examined. Collectively, these findings suggest that interactions between acidic casein-derived milk components and the biological surfaces involved in bacterial adhesion to S-HA result in an inhibitory effect that is selective for the oral streptococci examined.