Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DESCRIPTION
The present invention relates to a method of preparing
phosphorylated peptides from casein hydrolyzates which produces
a mixture of soluble peptides with a high degree of purity,
containing a high percentage of phosphoserine.
It is known that ~ milk casein and its derivatives contain
phosphoserines which impart valuable physical, chemical,
technological and physiological properties to the peptides
present therein.
Some authors have shown that intestinal absorption of calcium
increases as a result of the administration of casein in the rat
and that the phosphopeptides of casein are responsible for this
effect (Y.S. Lee, D. Noguchi and H. Naito, Brit. J. Nutr. 49,
67, 1983) .
Casein., phosphopeptides also increase calcium absorption in
normal chickens and those suffering from rickets (H. M. Mykkanen
and R.H. Wasserman, J. Nutr. 110, 2141, 1980).
It is therefore of interest to obtain casein phosphopeptides in
substantially pure form by simple and inexpensive methods.
The methods used up to now for the purification of
phosphopeptides from tryptic casein hydrolyzates provide for
precipitation with the use of a calcium or Fe3+ salt (EP-A-0 090
406 and JP-59-159793) or passage over activated carbon or over
cation-exchange resin (JP-59-159792).
It is known that aluminium (A1203) produced by thermal
dehydration of aluminium hydroxide, in the form of a powdery,
porous material, generally in particles of from 40 to 250 mesh,
can be used for the specific immobilization of phosphoric acid
(R-P03H2) and as a substrate for rendering phosphorylated
biomolecules insoluble in . accordance with affinity
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chromatography principles; these uses have been described and
discussed by Coletti-Previero and Previero (Analyt. Biochem.
180, 1-10, 1989).
EP-A-0 218 506 describes a method for the adsorption of
monosubstituted derivatives of phosphoric acid on alumina and
their subsequent elution by means of a phosphate buffer. The
specific applications comprise the fixing of phosphoderivatives
on alumina at ambient temperature and at a pH of between 2.0 and
8.5 in order to isolate and determine these phosphoderivatives
in biological media (for example, to render phosphorylated
enzymes insoluble).
It has been found that alumina can advantageously be used for
the purification of phosphopeptides from casein hydrolyzates,
producing a mixture of soluble peptides with a high degree of
purity, containing a large quantity of phosphoserine.
A first subject of the invention is therefore a method for the
selective purification of phosphopeptides from hydrolyzed
casein, characterized in that it comprises adsorption of the
casein hydrolyzate on alumina and elution of the selectively-
adsorbed phosphopeptides with ammonium hydroxide.
Another subject of the invention is a mixture of purified
phosphopeptides which can be produced by the above-mentioned
selective adsorption and elution method.
The method according to the invention has considerable
advantages such as speed, a good purification yield, and an
absence of contaminants in the peptides obtained. The use of
ammonium hydroxide for the elution of the phosphopeptides
constitutes a further improvement since it avoids contaminating
the peptides with the phosphorus contained in the buffer and the
eluent, and because the eluent can easily be removed in the form
of ammonia in the subsequent peptide lyophilization process.
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The peptides obtained by the method of the invention are soluble
in water and generally have a phosphoserine content equal to at
least 25~ of the total amino-acids.
The mixture of phosphopeptides obtained by the method according
to the invention can advantageously be used for the preparation
of pharmaceutical or dietary compositions for promoting the
absorption of metal ions such as calcium, gold, iron, lithium,
magnesium and zinc in the human organism; the purified
phosphopeptides can also be used for ionography purposes.
The phosphopeptides can also be used in pharmaceutical
formulations for anti-caries treatment. The pharmaceutical or
dietary compositions preferably comprise the~phosphopeptides in
association with lactulose and/or lactose.
The substrate on which the method according to the invention is
performed is constituted by a casein hydrolyzate obtainable by
enzymatic or chemical hydrolysis. The enzymatic hydrolysis
method's are known per se and preferably involve the use of
proteolytic enzymes which simulate the protein digestion which
takes place in vivo in the human body. For example, it is
possible to use pancreatin which is a complex mixture containing
trypsin, chymotrypsin and other secondary proteolytic enzymes.
The use of casein hydrolyzates with trypsin or mixtures of oc-
chymotrypsin and trypsin is particularly preferred.
The hydrolyzates are obtained by incubation of casein with
proteolytic enzymes at pH 8.5 and 37°C (see SIGMA catalogue,
1998). lkg of casein gives approximately 1kg of hydrolyzate from
which approximately 150 g of purified phosphopeptides is
obtained.
The hydrolyzates produced by chemical methods are obtained by
hydrolysis with 1N hydrochloric acid at 100°C under reflux.
' ~ _ ,~ ' , , . ,
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The alumina used in the method according to the invention may be
any commercially available activated alumina. During the
adsorption on alumina the pH of the hydrolyzed product is
preferably regulated to values of between 6.0 and 7.5.
The ammonium hydroxide used is typically an aqueous solution,
preferably having an NH3 concentration of from 12 to 15$.
The methods for the preparation of phosphopeptides and their
analytical characterization are described in the following
examples.
Example 1 - Preparation of phosphopeptides from a tryptic
hydrolyzate
125g of tryptic casein hydrolyzate was dissolved in 1.251 of
distilled H20, the pH being adjusted to 7.0 at 23°C. 100g of
activated alumina was added to the solution and gentle stirring
of the suspension was maintained. The pH, which tended to
increase, was readjusted to 7.0 with hydrochloric acid.
After 20 minutes, the solid phase was separated by filtration,
washed with distilled water and re-suspended in 150m1 of
distilled water; the pH was then brought to 9.5 with NH40H, with
constant stirring.
After 15 minutes, the mixture was filtered and the alumina was '
washed with 50m1 of distilled H20, the filtrate and washings
being collected and then lyophilized. The phosphopeptide
fraction was recovered as amorphous powder with a yield of 1.65g
and the remaining traces of NH3 were eliminated under vacuum in
the presence of concentrated sulphuric acid.
Example 2 - Preparation of phosphopeptides from an acid
hydrolyzate
Alternatively, 50g of casein was suspended in 500m1 of 1N HC1 in
a flask with a reflux condenser with heating to 100°C in a bain
.
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marie, conditions of incomplete boiling being maintained to
prevent dispersion of the solid.
After 150 minutes, the mixture was filtered and the filtrate was
brought to pH 6. 50g of neutral activated alumina was added.
After the mixture had been stirred slowly for 30 minutes, the
alumina was collected by Buchner, washed with water and re-
suspended in 80m1 of water. The pH was brought to 9.5 with
concentrated NH40H and the mixture was stirred slowly for one
hour.
The liquid phase was collected by filtration, washed with 40m1
of water and lyophilized. The lyophilizate obtained consisted
of 2g of phosphopeptides with discrete chromatographic
homogeneity, that is, constituted by a mixture of two-three
products.
Analytical characterization of the natural phosphopeptides
The following tests were carried out with the phosphopeptides
obtained from the tryptic hydrolyzate, for the sole reason that,
in this case, it is possible to use the starting material, which
is soluble, as a control. The characteristics of the products
obtained by the two systems, that is, by enzymatic or chemical
hydrolysis, are, however, similar.
The phosphopeptides obtained by the methods described above
constituted, as stated, a discretely homogeneous material
composed mainly of dipeptides containing Glu and P-Ser. To
demonstrate this, high-voltage electrophoresis was performed on
20~ polyacrylamide gel.
Tests carried by the conventional Laemmli method as described by
Donella-Deana et al (Bioch. Bioph. Res. Comm., 106, 1309, 1989)
did not succeed in identifying the phosphopeptides which clearly
had molecular weights of less than 1000.
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The amino-acid composition of the phosphopeptides was also
determined; all contained P-serine and the organic phosphorus
determination showed the phosphorus enrichment brought about by
the purification method.
Analysis of the amino-acids (as ~ of the total amino-acids)
Starting hydrolyzate Phosphopeptide fraction
Glu 23.66 25.06
Ser 7.54 25.76
As can be seen, the purified phosphopeptide fraction contained a
clearly greater percentage of serine. The content of other
amino-acids varied from 1.8 to 6~, approximately.
Organic phosphorus determination
The following values were obtained (mean ~ SD of four tests) with
the use of Martin and Doty's method (Anal. Chem., 21, 965, 1949)
with silicon tungstate as the precipitant and ammonium molybdate
as the specific reagent:
Phosphate (~..~.g/mg of casein)
Starting hydrolyzate 0.73 ~ 0.05
Phosphopeptide fraction 4.30 ~ 0.35
Interaction with metal salts
The chelation capacity for calcium was determined by Ca-specific
electrode (Metrohm, calomel reference electrode). The affinity
(1/K). of the purified phosphopeptides for calcium was
approximately twice that of the hydrolyzate:
Kphosph. 1.7 mg/ml
Khydrol. 3. 6 mg/ml.
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The chelation capacity for iron and gold ions was investigated
in solution by a conductometric method, by means of a Radiometer
CDM3 conductivity meter.
Solutions of FeCl3 and of AuCl4H in double-distilled H20 were
used at concentrations of from 0.2 to 1.4 mM and the
phosphopeptides were used at concentrations of from 0.5 to 250
mg/ml.
The relative affinities of the purified phosphopeptides for Fe
and Au were, respectively:
Fe 1.1 mg/ml
Au 1.2 mg/ml.
Similar chelation properties were found for zinc. The results
obtained are summarized in Tables 1 and 2.
TABLE 1
Results obtained with the Ca-specific electrode, expressed as
percentage of Ca chelated in dependence on increasing
concentrations of starting tryptic peptones or of purified
phosphopeptides
Concentration (mg/ml) Tryptic peptones Phosphopeptides
15.0 98 100
12.5 90 98
10.0 88 94
7.5 74 88
5.0 62 82
2.5 40 63
Values of the dissociation constants for the following
equilibria can be obtained from this data by a graphical method:
Peptones + Ca ---> peptones-Ca
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phosphopeptides + Ca ---> Phosphopeptides-Ca
TABLE 2
Results of chelation tests performed by a conductometric method
by mixing increasing quantities of (Ca, Fe, Au) ions with a
fixed quantity of phosphopeptides (250 mg/ml) in a final volume
of 50 ml of double-distilled water.
The quantity of ions added (in ~.1 of 1 M solutions) can be
compared with the conductivity values, expressed in ~s.
Subtraction of the conductivity relating to the phosphopeptides
alone (F) from that relating to the phosphopeptide-ion mixture
gives values which differ to a greater extent from those
relating the ions alone the greater is the degree of chelation.
Quantity of Ca Conductivity Conductivity Conductivity
Ca (F+Ca) - F (F+Ca)
0.0000 1.2000 0.0000 100.00
10.000 38.000 20.000 120.00
20.000 73.000 50.000 150.00
30.000 115.00 90.000 190.00
40.000 150.00 125.00 225.00
50.000 190.00 150.00 250.00
Quantity of Ca Conductivity Conductivity Conductivity
Ca (F+Ca) - F (F+Ca)
60.000 225.00 185.00 285.00
70.000 260.00 210.00 310.00
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Quantity ConductivityConductivityConductivi~y
o Fo
Fe (FTFe) - (FTFe)
F
0.0000 1.2000 0.0000 100.00
10.000 90.000 0.0000 90.000
20.000 200.00 50.000 150.00
30.000 260.00 150.00 250.00
40_000 360.00 250.00 350.00
50.000 450.00 350.00 450.00
60.000 500.00 400.00 500.00
70.000 600.00 500.00 600.00
Quantity ConductivityConductivityConductivity
of Au
Au (F+Au) - (F+Au)
F
0.0000 1.2000 0.0000 100.00
10.000 80.000 0.0000 90.000
20.000 210.00 15.000 115.00
30.000 300.00 100.00 200.00
40.000 410.00 190.00 290.00
50.000 470.00 270.00 370.00
60.000 550.00 325.00 425.00
70_000 610.00 420.00 520.00
To demonstrate that the chelation capacity of the
phosphopeptides of the invention is not an aspecific effect, a
comparison test was carried ouL with the use of non-
phosphorylated peptides such as glycyl glycine and enkephaline.
In the case of enkephaline and Qlycyl Qlycine, the values
obtained as the difference of (peptide + ion) - peptide coincide
with those relating to the conductivity of the ion alone.
This means that no chelation of Ca, of Fe, or of Au was evident
in the conditions used.
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Another subject of the invention is therefore constituted by the
use of the peptides which can be produced by the above-described
method to facilitate intestinal absorption of metal ions such as
calcium, iron, lithium, magnesium, gold and zinc.
Effect of phosphopeptides on the uptake of 45Ca "per os" in the
rat
Twelve male Wistar rats (350g body weight) were divided into
three groups and housed for three days in cages for two with
food "ad libitum". A quantity of 16.6~,Ci of 45Ca (+ 1mM Ca cold;
approximately 7x106 cpm) was introduced into the bottles
containing the drinking water and the quantity of water drunk in
the course of three days (about 300 ml) was measured at the end
of the test, when the rats were killed by decapitation. The
whole blood (5 ml), heparinized, was deproteinized with 10~
final trichloroacetic acid and the residual radioactivity was
determined by scintillation.
The radioactivity values (mean ~ SD of four determinations) were
as follows:
Groups Radioactivity (cpm/rat)
Control 683 ~ 68
+,.1 g hydrolyzate/500 ml 640 ~ 55
+ 1 g phosphopeptides/500 ml 2240 ~ 98
As can be seen, the amount of calcium absorbed by the rats was
increased more than three times by the phosphopeptides and
remained unchanged by the hydrolyzate.
Effect of phosphopeptides on intestinal absorption of calcium in
man
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Six healthy volunteers ingested lg of calcium gluconate (Sandoz)
and their urine was collected for 24 hours. After a "wash out"
period of 48 hours, they ingested lg of calcium plus 2.8g of
phosphopeptides dissolved in 200m1 of water and their urine was
collected for 24 hours.
Urinary excretion of calcium was then evaluated as shown in
Table 3.
TABLE 3
Urinary excretion of calcium in man (m = male; f = female)
Subjects Calcium (mmo1/die)Calcium+phosphopeptides'(mmol/die)
L.G. m 7.50 8.50 colorimet. met.
(58)
(arsenazo II)
M.B. f 1.51 1.40
(56)
A.L. m 7.48 9.43
(45)
G.M. f 7.14 mean=5.69 6.95 mean=6.4
(41)
S.B. f 3.71 4.92
(35)
M.G. m 6.80 7.20
(51)
The peptide L-Glu-L-Ser-P was synthesized chemically and it was
found that the effects of the natural peptides were reproduced
faithfully by the synthetic peptide.
