The Emerging Role of Dairy Proteins and Bioactive

Peptides in Nutrition and Health

Bioactive Properties of Milk Proteins with Particular Focus

on Anticariogenesis1
William R. Aimutis2
Food Technical Development Center, Cargill, Inc., Wayzata, MN 55391

ABSTRACT Beyond nutrition, there is an increasing amount of data and information to demonstrate a bioactive role
for dairy components in adults including a role in prevention of dental caries. Specifically, the casein fraction and
hydrolysates thereof have been the focus of researchers investigating cariogenicity prevention. Tooth enamel is a
polymeric substance consisting of crystalline calcium phosphate embedded in a protein matrix. Dental caries develop
by acidic demineralization (calcium and phosphorus solubilization) of tooth enamel. Demineralization occurs directly
(acidic food consumption) or indirectly (by fermentation products of dental plaque odontopathogenic bacteria growing
on residual food particles between teeth or adhering to the plaque). Research efforts with milk derived bioactive
peptides have focused on inhibition of cariogenic, plaque-forming bacteria, inhibition of tooth enamel demineralization,
and subsequent enamel remineralization. Caseinophosphopeptides (CPP) and glycomacropeptide (GMP) have been
patented for use in common personal hygiene products to prevent dental caries. Research has shown CPP and GMP
to be growth inhibitory to the cariogenic bacteria Streptococcus mutans and other species. Additionally, CPP forms
nanoclusters with amorphous calcium phosphate (AMP) at the tooth surface to provide a reservoir of calcium and
phosphate ions to maintain a state of super saturation with respect to tooth enamel. This would buffer plaque pH, and
also provide ions for tooth enamel remineralization. Glycosidic structures attached to GMP are important to numerous
bioactive properties of the peptide including anticariogenicity. Like CPP, GMP has shown inhibitory activity to enamel
demineralization and promotes tooth enamel remineralization. J. Nutr. 134: 989S–995S, 2004.

KEY WORDS: ● milk ● bioactives ● anticariogenicity ● caseinophosphopeptide ● glycomacropeptide

Dental caries (tooth decay) are a major public health problem
that plagues all countries in the world. Industrialized nations have
controlled the problem with fluoride enriched water and personal
hygiene products since early in the 1960s, but cariogenicity re-
mains a crisis that economically burdens the health care system to
an extent greater than many publicized diseases such as heart
disease, cancer, and hypertension. Dental disease remains a “si-
lent epidemic” in the United States that threatens children and
adults (1). As developing countries begin consuming more de-
veloped foods, tooth decay also is becoming an issue (2). High
risk of dental caries is accentuated by a number of sociodemo-
graphic variables including ethnicity and low socioeconomic sta-
tus. Certain individuals are also at risk as a complication of other
disease states including diabetes (3,4), obesity (5), and osteopo-
rosis (6). Researchers and product developers continue to search
for products to reduce overall severity and prevalence of dental
1
Published in a supplement to The Journal of Nutrition. Presented as part of
the 94th American Oil Chemists’ Annual Meeting & Expo held in Kansas City, MO,
May 4 –7, 2003. This symposium was sponsored by the National Dairy Council,
Kraft Foods Inc., The Whey Protein Institute, and the U.S. Dairy Export Council.
Guest editors for the supplement publication were Peter J. Huth, National Dairy
Council, Rosemont, IL; Donald K. Layman, University of Illinois, Urbana, IL; and
Peter H. Brown, Kraft Foods Research and Development, Kraft Foods Inc.,
Glenview, IL.
2
To whom correspondence should be addressed.
E-mail: bill_aimutis@cargill.com.
caries. A major emphasis has been placed on developing products
that are convenient to the consumer such as chewing gums and
sugar-free confections that offer a degree of protection from the
causative agents of tooth decay.
Milk is an excellent protein food that provides essential amino
acids and organic nitrogen for humans and animals of all ages.
Milk also contains factors that have anticariogenic properties:
calcium, phosphate, casein, and lipids. Dairy products were rec-
ognized in the late 1950s as a food group that is effective in
preventing dental caries. Shaw et al. (7) observed that milk, ice
cream, and cheese lowered incidence of dental caries in rats.
Epidemiological studies in recent years indicate children (8) and
adolescents (9) with low incidence of dental caries drank more
milk than those with high caries incidence. Elderly people that
eat cheese several times per week had a lower incidence of root
surface caries development (10). Several reviews describe the role
of milk and dairy products in dental caries prevention (11–13).
The purpose of this paper is to review the role of minor milk
proteins and bioactive peptides embedded in the major milk
proteins that inhibit cariogenicity.
Dental caries pathogenesis
Children’s teeth become infected with potential odonto-
pathogenic bacteria between middle of y 2 and end of y 3 of
life—the “window of infectivity” (14). Primary source of in-

0022-3166/04 $8.00 © 2004 American Society for Nutritional Sciences.

989S

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 990S
fection for infants is maternal, but certain environmental
conditions, such as infants born into a high caries-prone
population, can also favor nonfamilial infection (15). Chil-
dren that are not infected by a high maternal dose by 3 y of age
remain minimally colonized by odontopathogenic bacteria un-
til eruption of their secondary teeth.
Caries lesions, or tooth decay, are the clinical manifestation
of a pathogenic process that may have been occurring as a
series of interactions on the tooth surface for months or years.
Plaque is a biofilm over tooth enamel composed of viable and
nonviable bacteria, mucopolysaccharides, and other cellular
debris and metabolites. The first step in cariogenicity is that
indigenous oral bacteria begin decay by interacting with di-
etary constituents (e.g., sucrose) at the tooth enamel’s surface.
Dental plaque appearance on tooth enamel is the first overt
clinical evidence of this interaction. Plaque bacteria metabo-
lize dietary sugars to produce organic acids that solubilize tooth
enamel composed of hydroxyapatite crystals of calcium phos-
phate. When enamel is exposed to organic acids, solid calcium
phosphate is solubilized to free calcium that is removed from
the mouth by saliva movement. This process is termed demin-
eralization, but can be reversed by presence of salivary sodium
bicarbonate that aids in remineralization.
The role of bacteria in causing dental caries is a source of
continual controversy. At issue has been whether a specific
bacterial species or a nonspecific mixed bacterial flora is the
agent responsible. Also debated is if dental caries is an infec-
tious bacterial disease in the classical sense or an ecological
overgrowth (16). Frequent presence of Lactobacillus acidophilus
and Streptococcus mutans with caries activity gave credibility to
their being specific cariogens. However, many other indige-
nous oral bacteria are capable of producing substantial
amounts of organic acid from fermentable carbohydrates pro-
viding arguments for nonspecificity. Numerous studies have
shown some indigenous bacteria are capable of remineralizing
tooth enamel to prevent dental caries. To date, more research
in understanding mixed-bacterial ecology and metabolism is
needed to develop therapeutic strategies to counter excess acid
accumulation and tooth demineralization.
Dental caries is still the predominant cause of tooth loss in
all populations worldwide. Numerous approaches have been
used to protect children and adults from cavities. Milk and
dairy products have been identified as having cariostatic fac-
tors. However, milk-derived cariostatic factors have limited
effectiveness in their natural source because they would re-
quire large consumption of dairy products. Researchers have
focused on isolating protective factors from milk to use as food
additives or in personal hygiene products to reduce cariogen-
icity.
Milk proteins
Milk is synthesized in mammary secretory epithelial cells
and contains 2 major protein groups distinguished by their
solubility in unheated milk at pH 4.6 and 20°C: caseins
(insoluble) and whey proteins (soluble). Both groups have
unique physiochemical and biological properties. Caseins ac-
count for ⬃80% of the total protein in bovine milk, and exist
primarily as calcium phosphate stabilized micellular com-
plexes. Caseins are a heterogeneous family of proteins predom-
inated by ␣s1-, ␣s2-, ␤-, and ␬-caseins (17). Individual casein
proteins are small molecules with a molecular mass of 20 to 25
kDa, and primary amino acid sequences that are high in
proline content. Proline prevents casein molecules from hav-
ing much secondary structure (␣-helices, ␤-sheets, and
␤-turns). All caseins show genetic polymorphism and have
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SUPPLEMENT
post-translational modification with either phosphorus and/or
carbohydrate moieties. Caseins are relatively hydrophobic, but
have primary sequence clusters that have high surface hydro-
phobicity that contributes to functional properties such as
emulsification and foaming. Fox (18) and Wong et al. (19)
review the relationship of individual casein’s structure and
function.
Whey proteins (20% of total milk protein) are also a
heterogeneous, polymorphic group of proteins composed of
␣-lactalbumin (␣-LA,3 20%), ␤-lactoglobulin (␤-Lg, 50%),
serum albumin (BSA, 10%), immunoglobulins (10%), and
proteose peptones (⬍10%). Unlike caseins, whey proteins
have high levels of secondary, tertiary, and quaternary struc-
tures, and are typically heat-labile globular structures. All
whey proteins contain intermolecular disulphide bonds that
stabilize their structure. Whey proteins are not extensively
glycosylated, and none are phosphorylated. The dominant
proteins (␣-LA and ␤-Lg) are responsible for functional prop-
erties, predominantly foaming and gelation, that have been
commercialized in whey protein concentrate and isolate prod-
ucts.
Milk contains numerous minor proteins found mainly in
the whey and milk fat globule membrane fractions. These
minor proteins do not have significant functional properties
like casein and whey fractions, but many have been identified
as having physiological effects. The minor proteins include
enzymes, metal-binding proteins, enzyme inhibitors, vitamin-
binding proteins, and numerous growth factors (18). Several
minor dairy proteins have been included as bioactive ingredi-
ents in nutraceutical products.
Dairy protein bioactivities
Bioactive proteins and peptides are embedded in casein and
whey primary sequences. A variety of regulatory activities are
exerted by milk-derived bioactive sequences. Biological activ-
ities identified include modulators of digestive and gastroin-
testinal functions, hemodynamics (hypertension and gastric
blood flow), anticariogenicity, analgesic properties, growth
factors, immunoregulation, and nonimmune disease defense.
Most bioactivities are only expressed by peptides derived from
the amino acid sequence of native milk proteins. Digestive
proteases or in vitro proteolysis liberates bioactive peptides to
the host’s benefit. Milk-derived peptides are now commercially
produced and these peptides are being used as dietary supple-
mentation in functional foods and personal products. How-
ever, very few clinical trials have been done to prove safety of
milk-derived peptides. Most commercial companies have pre-
sumed safety on basis of a “safe” starting raw material. Acute
toxicity, allergenicity, and nutritional studies have not been
adequately conducted (20). Extensive reviews on milk-derived
bioactivities are written (21,22).
Lactoferrin is an iron-binding protein found in milk of
many species including bovine and human, and has been
observed to possess numerous bioactive properties. The bioac-
tive role of lactoferrin appears to be dependent on exceptional
iron-binding activity by the molecule. Iron availability from
an infant formula supplemented with bovine lactoferrin has
been evaluated by iron balance studies in human infants (23).
Iron retention was 36% in the supplemented group versus 28%
in the nonsupplemented group. Lactoferrin has also been
3
Abbreviations used: ACP, amorphous calcium phosphate; ␣-LA, alpha-
lactalbumin; ␤-Lg, beta-lactoglobulin; BSA, bovine serum albumin; CPP, case-
inophosphopeptides; GMP, glycomacropeptide; SCN⫺, thiocyanate; S-HA, sa-
liva-coated hydroxyapatite beads.
 MILD PROTEIN ANTICARIOGENICITY
shown to have an effect on cell line growth by stimulating
DNA synthesis (24,25). Other researchers have reported lac-
toferrin inhibits proliferation of cancer cell lines in vitro
(26,27). Lactoferrin has antibacterial activity toward Gram-
negative bacteria including the dental cariogen Streptococcus
mutans (28). Lactoferrin is a moonlighting protein that exhib-
its multiple functions that are expressed in different loca-
tions and at different times as extensively reviewed by
Shimazaki (29).
Other minor proteins in bovine milk have antibacterial
activity. Lysozyme is an effective antibacterial enzyme isolated
from milk, tears, and saliva. Human milk is a better source of
lysozyme (2.5 mg/100 mL) than bovine milk (0.025 mg/100
mL). The mechanism of action for lysozyme is to hydrolyze
␤(134)-glucosidic linkages in bacterial cell wall peptidogly-
can. Lactoperoxidase is a porphrin-containing peroxidase se-
creted by the mammary gland. This enzyme is part of a com-
plex that ultimately has antibacterial effects. Lactoperoxidase
catalyzes the oxidation of thiocyanate (SCN⫺) to hypothio-
cyanate by using hydrogen peroxide produced by endogenous
bacteria (Fig. 1).
Induced bioactive components are defined as activities de-
rived by proteolytic or immunological means (30). Protein
modification by proteases can occur in situ in the mammary
gland or digestive tract, or by in vitro digestion with commer-
cial proteases. The resulting peptides influence particular phys-
iological or metabolic processes. Immunological derived bio-
active components result from vaccinating cows to induce
production of antibodies to the vaccinate antigen.
Bovine ␬-casein treated with chymosin is cleaved into 2
peptides during the cheese making process. Para ␬-casein
(residues 1–105) remains with the curd, while a unique pep-
tide, glycomacropeptide (residues 106 –169), elutes with the
cheese whey (17). Glycomacropeptide (GMP) is a major com-
ponent of cheese whey protein as it is 15–20% of the total
protein. Glycomacropeptide is a glycophosphopeptide with no
aromatic amino acids. This could have positive implications
for certain dietary restricted populations.
Glycosidic structures (Table 1) attached to the peptide are
important in GMP bioactivity. The ability to bind enterotox-
ins from Vibrio cholerae and Escherichia coli has been reported
with GMP (31). Carbohydrates attached to GMP mimic re-
ceptor sites for enterotoxins. Therefore, in animal challenge
studies GMP binds enterotoxin and the complex is washed
from the intestinal tract. GMP treated with neuraminidase to
remove sialic acid carbohydrate moiety from the terminal end
of the glycosidic side chain on GMP lost its bioactivity.
Feeding mice GMP (1 mg/d) during in vivo trials protected the
animals from E. coli and V. cholerae enterotoxin associated
diarrhea (32).
Kawasaki et al. (33) demonstrated that GMP inhibits hem-
agglutination of 4 human influenza virus strains. The effect
was noted with low GMP concentrations (80 mg/L). Hemag-
glutination of Mycoplasma gallisepicum (34) and M. pneumoniae
(35) is not inhibited by GMP.
The role of GMP as a prebiotic is elusive. Prebiotics are
compounds that promote growth of desirable gastrointestinal
FIGURE 1 The mechanism of action for lactoperoxidase. Thio-
cyanante (SCN⫺) and hypothiocyanate (OSCN⫺).
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991S
TABLE 1
Glycomacropeptide glycosidic structure
Saccharide Composition and Structure
Monosaccharide GalNAc-O-Thr
Disaccharide Gal ␤133 GalNAc-O-Thr
Trisaccharide NeuAc ␣233 Gal ␤133 GalNAc-O-Thr
Trisaccharide Gal ␤133 (NeuAc ␣236)GalNAc-O-Thr
Tetrasaccharide NeuAc ␣233 Gal ␤133(NeuAc ␣236)
GalNAc-O-Thr
Galactose (Gal), N-acetyl galactosamine (GalNAc), neuraminic acid
(NeuNAc), oxygen (O), threonine (Thr).
bacteria (probiotics). Kehagias et al. (36) reported bifidobac-
teria growth was promoted by a casein fraction that was similar
to GMP. Poch and Bezkorovainy (37) demonstrated that
bovine casein and whey digest promoted bifidobacteria
growth. Petchow and Talbott (38) found that growth promot-
ing activity for some bifidobacteria species is present in bovine
milk ultrafiltration permeate and retentate. GMP would be
present in the retentate. Therefore, it appears permeate may
contain amino acids and other compounds also necessary for
bifidobacterial growth.
Milk micelles contain physiologically significant amounts
of calcium and phosphorus. Phosphorus in milk is bound via
monoester linkages to casein serine residues. The presence of
phosphorus and calcium bound to casein helps to maintain
thermodynamically stable casein micelles in fluid milk. Ca-
seinophosphopeptides (CPPs) are phosphorylated casein-de-
rived peptides produced by proteolytic digestion of ␣s1-, ␣s2-,
and ␤-casein in vitro or in the digestive tract (Table 2). CPP
complexes and solubilizes minerals, especially calcium. CPP
improves bioavailability in intestinal absorption by keeping
minerals soluble and preventing precipitation. Most CPPs
contain a serine phosphate cluster and glutamyl residues in the
sequence of 3 phosphoseryl groups followed by 2 glutamic acid
residues. These amino acids serve as mineral-binding sites
because of negatively charged side chains. However, there is a
difference in calcium binding activity depending on the casein
phosphopeptide source (39). The order of mineral chelating
ability is ␣s2-casein ⬎ ␣s1-casein ⬎ ␤-casein ⬎ ␬-casein.
Casein phosphopeptides released from casein molecules are
resistant to further proteolytic breakdown in the intestinal
TABLE 2
Primary sequences of caseinophosphopeptides derived
from ␣-caseins and ␤-caseins
-Casein (fragment) Sequence
␣s1-f(59–79) -gln-met-glu-ala-glu-ser(P)-ile-ser(P)-ser(P)-ser(P)-
glu-glu-ile-val-pro-asn-ser(P)-val-glu-gln-lys-
␣s1-f(112–119) -val-pro-asn-ser(P)-ala-glu-glu-arg-
␣s2-f(5–18) -glu-his-val-ser-ser(P)-ser(P)-glu-glu-ser-ile-ile-
ser(P)-gln-glu-
␣s2-f(29–34) -asn-pro-ser(P)-lys-glu-asn-
␣s2-f(55–64) -gly-ser(P)-ser(P)-ser(P)-glu-glu-ser(P)-ala-glu-val-
␣s2-f(127–147) -gln-leu-ser(P)-thr-ser(P)-glu-glu-asn-ser-lys-lys-
thr-val-asp-met-glu-ser(P)-thr-glu-val-phe-
␤-f(12–23) -ile-val-glu-ser(P)-ser(P)-ser(P)-glu-glu-ser-ile-lys-
(variant A)
␤-f(12–23) -ile-val-glu-ser(P)-lys-ser(P)-glu-glu-ser-ile-lys-
(variant D)
 992S
tract. Formation of CPP in the intestinal tract should increase
the concentration of soluble Ca available to the host animal
(40). However, conflicting data has been reported on Ca
absorption, Ca balance, and bone formation. Although CPPs
seem to have little effect in young, healthy animals (41– 43),
positive data have been observed in ovariectomized rats (44)
and postmenopausal women (45). Other studies showed Ca
and Zn absorption to be improved in rat pups (46) on oat- or
soy-based infant formulas and in healthy individuals (47) fed
rice-based or whole grain diets.
Lactoferricin B is an induced bioactive peptide encrypted
within lactoferrin, and can be liberated by the digestive en-
zyme pepsin (48). Lactoferricin is active against both Gram-
positive and Gram-negative bacteria, and is more bactericidal
than undigested lactoferrin because the peptide’s smaller size
may facilitate access to susceptible sites on the microbial
surface (49). The effect of Lactoferricin B against oral odon-
topathogenic bacteria has not been investigated.
Anticariogenic activity of milk bioactive proteins
Early studies recognized that dairy products (milk, casein,
caseinates, and cheeses) exhibited anti-caries activity (50,51).
Acid casein (insoluble) as an active ingredient in toothpaste
was effective at reducing dental caries, but was required at very
high levels for activity (52,53). Sodium caseinate solubilized in
water and fed to rats in a caries model was shown to be
anticariogenic (54). Sodium caseinate as an ingredient in a
chocolate confectionary reduced cariogenicity, but high levels
of caseinate (17%) were required to elicit an effect and the
product was unpalatable (55,56). Therefore, early studies dem-
onstrated that casein was an effective anticariogenic sub-
stance, but casein’s adverse organoleptic properties and the
large amount required for efficacy precluded its use as a food or
toothpaste.
Tryptic digestion of caseinate did not destroy the proteins’
ability to prevent enamel demineralization in a human oral
caries model (57). Analysis of human dental plaque samples
found elevated concentrations of casein peptides, calcium, and
phosphorus. It was concluded that these peptides were casein-
ophosphopeptides derived from specific tryptic activity on
␣s1-, ␣s2-, and ␤-caseins. This prompted investigators to focus
on casein peptides in subsequent research.
Many of the reported physiologically active or bioactive
components account for a very minor fraction of total milk
constituency. This group includes lactoferrin, lysozyme, lac-
toperoxidase, folate-binding protein, growth factors, and other
proteins (Table 3). Immunoglobulin proteins also have a
protective role in vivo. Bioactive components that have a role
in prevention of dental caries would need to mechanistically
inhibit odontopathogenic bacteria and/or bind minerals.
Prevention of dental caries by milk-derived bioactive pep-
tides is a complex physical and chemical sequence of cascading
events. In general, bioactive peptides with anticariogenic ac-
tivity have multiple functions to prevent dental lesions includ-
ing bacterial inhibition, competitive exclusion to enamel
binding sites, improved buffering capacity in the pellicle sur-
rounding teeth, reduced enamel demineralization, and enamel
remineralization. Anticariogenicity studies with dairy bioac-
tive peptides have been accomplished with a number of in
vitro, in situ, and in vivo model systems. Much of the work has
been done with caseinophosphopeptides-colloidal amorphous
calcium phosphate (CPP-ACP). Caseinophosphopeptides in-
hibit dental caries lesions by influencing the demineralization/
remineralization process of dental enamel. A 1% (wt:v) CPP
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TABLE 3
Bioactive proteins secreted in bovine milk
Protective Proteins Hormones
Immunoglobulins Thyrotropin releasing
Proteose peptones hormone (TRH)
Lactoferrin Somatostatin (SIH)
Transferrin Calcitonin
Insulin
Growth Factors Relaxin
Epidermal growth factor (EGF) Thyroid stimulating hormone
Tissue growth factor ␤ (TGF␤) (TSH)
Insulin-like growth factor (IGF-1) Luteinizing releasing
hormone (LRH)
Enzymes Gastrin releasing peptide
Lactoperoxidase (GRP)
Lysozyme Adrenocorticotropichormone
Plasmin (ACTH)
Xanthine oxidase Prolactin
Glucose oxidase
solution can stabilize 60 mmol/L CaCl2 and 36 mmol/L sodium
phosphate at pH 7.0 to form CPP-ACP complexes (58).
Specific-pathogen free rats orally infected with Streptococ-
cus sobrinus had a reduced incidence of smooth surface caries
after CPP-ACP solutions were applied to the animal’s teeth
twice daily. A dose dependent response was observed with a
0.1% (wt:v) CPP-ACP producing a 14% reduction, and 1.0%
CPP-ACP a 55% reduction relative to a distilled water con-
trol. These results were similar to a 50 ppm fluoride control. A
synergistic effect was reported when CPP-ACP (0.5%) and
fluoride (500 ppm) were applied together to rat’s teeth. Non-
phosphorylated casein peptides had no anticariogenic effects.
A synthetic octapeptide, Ac-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-
Ser(P)-Glu-Glu-NHMe, significantly reduced caries activity
in the rat model. This confirmed that the xn-Ser(P)-Ser(P)-
Ser(P)-Glu-Glu-xn portions of CPP are associated with anti-
cariogenicity. However, the synthetic octapeptide was not as
effective in binding ACP or in anticariogenicity as CPP-ACP.
This indicates other residues and/or conformational specificity
such as in the longer ␣s1- and ␤-peptides are required for full
activity (58).
A human in situ caries model has been used to study the
ability of 1% CPP-ACP to prevent enamel demineralization
(59). Humans are fitted with a removable dental appliance
that contains a left and a right pair of enamel slabs placed in
the mouth to produce a plaque retention site. Frequent expo-
sure to sucrose solutions elevated levels of oral mutans strep-
tococci and lactobacilli. Subsurface enamel demineralization
resulted in an incipient “caries-like” lesion. Two daily expo-
sures of CPP-ACP solution to 1 side of the enamel slab
reduced (51 ⫾ 19%) enamel mineral loss compared to the
control side. Plaque exposed to CPP-ACP had 2.5 times more
Ca and phosphorus than control plaque. Caseinophosphopep-
tides stabilize ACP; in turn this is used to localize ACP in
dental plaque, provides a large calcium reservoir within
plaque, and slows diffusion of free calcium (60). Restricted
mineral loss during a cariogenic episode is likely to occur, and
conversely provide a source of calcium for remineralization.
Therefore, the mechanism of anticariogenicity for CPP-ACP
is that this bioactive peptide substantially increases the level of
amorphous calcium phosphate in plaque depressing enamel
demineralization and enhancing remineralization. As the con-
centration of CPP-ACP in contact with tooth enamel in-
creases so does remineralization (Fig. 2). An increased con-
 MILD PROTEIN ANTICARIOGENICITY
FIGURE 2 Dose response of CPP on remineralization of tooth
enamel subsurface lesions (61).
centration of free and bound calcium in 0.5 and 1.0% CPP-
ACP preparations was most effective at remineralization (61).
Sodium caseinate, CPP, and GMP inhibited adherence of
oral bacteria to saliva-coated hydroxyapatite beads (S-HA)
(62). The potential dental pathogens, Streptococcus sobrinus
OMZ 176 and Streptococcus sangius OMZ 9, were competi-
tively excluded from S-HA beads by casein derivatives. How-
ever, Actinomyces viscosus Ny 1 was not excluded from the
S-HA beads. Another experiment in this study demonstrated
that caseinate, CPP, and GMP were capable of binding di-
rectly to bacterial cell walls of the examined strains. These
results offer another possible mechanism for anticariogenicity
by dairy-derived bioactives. By selectively inhibiting strepto-
coccal adhesion to teeth, dairy bioactives could modulate the
microbial composition of dental plaque and favor establish-
ment of less cariogenic species such as oral actinomyces (62).
This could also control acid formation (buffering) in dental
plaque, in turn reducing hydroxyapatite dissolution from tooth
enamel (63).
Milk components CPP and GMP incorporated into the
salivary pellicle, and reduced adherence of S. sobrinus and S.
mutans (64). Electron microscopy visualized GMP on the
pellicle surface in a globular-micellular form. Conversely, CPP
was found in microglobular and geometrically irregular struc-
tures. This relatively selective inhibition of S. mutans could
eventually produce a noncariogenic plaque. Additional evi-
dence was reported by Rose (60) indicating CPP-ACP would
compete with calcium for plaque Ca binding sites. This will
reduce the amount of calcium bridging between the pellicle
and adhering cells and between cells themselves. High extra-
cellular free calcium concentrations could exist that have
bacteriostatic or bacteriocidal effects on odontopathogenic
bacteria (65).
Schupbach et al. (64) demonstrated GMP could prevent
cariogenic bacterial adhesion in an in vitro model. The re-
searchers speculate GMP reduces dental caries by changing the
microbial population of dental plaque from 1 predominated by
Streptococcus mutans and S. sangius to a less cariogenic popu-
lation predominated by Actinomyces viscous.
Whey proteins from cottage cheese manufacture or acid
casein precipitation were not as effective as CPP in preventing
Ca and phosphate solubilization from hydroxyapatite (66).
Although whey proteins may not prevent enamel demineral-
ization, it has been suggested whey may exert a topical anti-
cariogenic effect by acting as a buffer (54). Minor whey pro-
teins may provide anticariogenic effect when they are enriched
or purified from the heterogeneous whey protein mixture. For
example, lactoperoxidase and lysozyme restricts S. mutans me-
tabolism (67). Lactoferrin inhibits adherence of S. mutans to
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saliva-coated hydroxyapatite beads (68). Lactoperoxidase and
lysozyme synergistically work together to inhibit glucose me-
tabolism by S. mutans thereby reducing acid production in the
plaque environment (69). Proteose peptone fractions 3 and 5
were shown to inhibit demineralization of hydroxyapatite in
vitro (70). Both proteose peptone fractions are embedded
within ␤-casein and are liberated by plasmin hydrolysis
(71,72). Plasmin is an endogenous protease of milk.
Commercialization of milk bioactives
There is commercial interest in production of dairy-derived
bioactive peptides with the purpose of using them in commer-
cial products such as toothpastes, gels, or mouth rinses. Casein
GMP was patented as an antimicrobial agent effective against
S. mutans (73,74). Product developers incorporated GMP into
commercial products that were protected by patents (75). The
uniqueness of this patent was the use of hydrolyzed gelatin as
a stabilizer in the compositions. A patent was issued (76) for a
multicomponent anticaries dentifrice composition and meth-
ods to use that contained GMP and fluoride in combination.
Another patent (77) was issued when it was disclosed that
GMP in combination with xylitol would act synergistically to
yield enamel remineralization results greater than those ob-
served with xylitol and fluoride in combination (Fig. 3). These
patent’s claims were supported by in vitro enamel remineral-
ization data. A patent has been filed (Warner-Lambert Com-
pany) to use CPP in chewing gum compositions to promote
anticariogenicity. Lactoperoxidase and lactoferrin have been
formulated into toothpaste to be bactericidal to cariogenic
bacteria. Glucose oxidase in this toothpaste activates the
lactoperoxidase system to produce hypothiocyanous acid and
hypothiocyanite ions in the oral environment (Fig. 4), result-
ing in inhibition of odontopathogenic bacteria.
Future research
Tooth decay is a series of interactions that occur on a tooth
surface, and remains a critical health problem despite advances
made in fluoridation of water and toothpastes. Research data
outlined in this paper presents a strong case that dairy-derived
bioactive peptides reduce dental caries in humans using in situ
FIGURE 3 Synergistic effects of glycomacropeptide-containing
and xylitol-containing toothpastes on tooth enamel remineralization in
situ. Healthy adult subjects were fitted with palatal retainers that had
demineralized tooth enamel planks. Subjects brushed their teeth with
toothpastes that contained the active ingredients 10%-glycomac-
ropeptide (GMP), 10%-xylitol (Xylitol), or 10%-glycomacropeptide and
10%-xylitol (GMP/Xyl). The placebo toothpaste contained silica abra-
sive without fluoride. Hydroxyapatite thickness was determined by
radiography.
 994S SUPPLEMENT
FIGURE 4 The effectiveness of tooth brushing for 1 min with a
commercial toothpaste that contains glucose oxidase (10,000 U), lac-
toperoxidase (15,000 U), and lysozyme (16 mg) as active ingredients on
the generation and decomposition of hypothiocyanous acid (HOSCN)
and hypothiocyanite ions (OSCN⫺) (69).
methods. However, future work should be focused on in vivo
and epidemiological effects of bioactive consumption in re-
ducing or eliminating dental caries wherein artificial plaque-
developing environments are not depended upon to study
caries prevention. Minimally, an effect should be observed in
reducing caries severity. Products developed with CPP to date
have enabled the peptides to remain in prolonged contact
(such as chewing gum and toothpastes) with the plaque envi-
ronment to prevent dental caries. The primary mechanisms of
action are a reduction in enamel demineralization and im-
proved enamel remineralization.
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