Method for separation and concentration of phosphopeptides

The present invention relates to a method for separating and concentrating acidic peptide, especially phosphopeptide having a phosphoserine residue, from a peptide mixture prepared by digesting casein with protease.

FIELD OF THE INVENTION 
The present invention relates to a method for separating and concentrating 
acidic peptides, especially phosphopeptide having a phosphoserine residue, 
from a peptide mixture prepared by digesting casein with protease. 
More particularly, the present invention relates to a method for separation 
and concentration of these peptides either without using any organic 
solvent such as ethyl alcohol, pyridine, etc., or without adding calcium, 
iron, etc. In addition, the present invention is also applicable not only 
to trypsin hydrolysates of casein which are casein phosphopeptides 
(hereafter referred to as CPP) generally termed as plural kinds of 
peptides containing a phosphoserine residue, but also widely to 
hydrolysates with proteases other than trypsin. 
PRIOR ART 
Several methods were established around 1970 for preparing casein-derived 
phosphopeptides on a laboratory scale 
For example, W. Manson and W. D. Annan precipitate and fractionate 
phosphopeptides by adding barium chloride and ethyl alcohol to a soluble 
fraction of pH 4.6 in the solution obtained by cleaving .beta.-casein with 
trypsin at pH 8 at 20.degree. C. (Arch. Biochem., 145, 15-26 (1971)). 
On the other hand, Naito and Suzuki roughly fractionate a solution of 
trypsin digestion products of .beta.-casein by gel filtration, pass the 
fraction through an ion exchanger (DOWEX 50-X2) column and then carry out 
the elution with pyridine-acetate buffer (pH 2.5, 0.2N pyridine) to 
fractionate phosphopeptides, etc. (Arg. Biol. Chem., 38 (8), 1543-1545 
(1974)). 
Also West adjusts the pH of a solution of .alpha.-casein treated with 
trypsin to 2 to remove the precipitates, then applies the filtrate to gel 
filtration (Sephadex G-25M), collects a large molecular fraction eluted in 
Void Volume and then passes the fraction through an anionic exchanger 
(DEAE Sepharose A50) to adsorb phosphopeptides to the anionic exchanger. 
However, these methods are basically to prepare CPP for the purpose of 
research or to confirm the presence or purity of CPP. In order to 
industrialize these methods as preparing food materials, inappropriate 
reagents such as barium chloride, pyridine, etc. are used, or operations 
which are difficult for scaling up for industrialization are involved. 
On the other hand, for the purpose of preparation on a large scale, it has 
been proposed to separate phosphopeptides from other peptides through an 
ultrafiltration membrane, utilizing the property of phosphopeptides that 
phosphopeptides from peptides formed by decomposition of casein with 
protease do not precipitate but from a large aggregate by the addition of 
minerals such as calcium (Japanese Patent Application Laid-Open No. 
123921/81). 
Furthermore, it is known to precipitate and isolate phosphopeptides by 
adding calcium and ethyl alcohol or by adding ferric chloride to the 
cleavage products of casein with trypsin (Japanese Patent Application 
Laid-Open Nos. 170440/83 and 159793/84). 
PROBLEMS TO BE SOLVED BY THE INVENTION 
In order to use phosphopeptides as food materials or medical materials, 
incorporated of barium salt should be avoided. Therefore, the method of 
manson and Annan using barium chloride in the prior art described above is 
not suitable. 
Furthermore, the method using ethyl alcohol requires large quantities of 
expensive ethyl alcohol. In the case of the treatment on a large scale, 
problems are, thus, encountered especially from economic and safety 
considerations. In addition, since CPP is also known to be a calcium 
absorption accelerator (British J. of Nutrition, 43, 457-467 (1980)), 
minerals such as calcium and the like are used in combination separately 
from other sources, where phosphopeptides are used in food materials as 
the calcium absorption accelerator; therefore, it is preferred that 
minerals such as calcium are not contained in phosphopeptides. In such a 
case, it is recommendable to supply phosphopeptides in the market in a 
calcium-free or iron-free state, because its application area may be 
broadened and hence its commercial value becomes high. 
On the other hand, the laboratory scale methods described above involve 
various problems. Firstly, the method using the barium salt is 
inapplicable to food materials. Turning next to the method using gel 
filtration in combination with ion exchange column chromatography, it is 
impossible to apply gel filtration to a large scale process at this time. 
Further in the latter method, anionic exchange resin principally has 
affinity for phosphopeptides having a phosphoserine residue. However, 
casein itself is an acidic protein and protease digestion products also 
keep affinity for anionic exchanger as a whole. Therefore, in order to 
isolate phosphopeptides from the whole peptides, severe selectivity is 
required. In actuality, when used singly, most anionic exchange resins 
have an extremely weak adsorption to phosphopeptides. Even though an 
anionic exchange resin having affinity for a large molecular substance 
such as phosphopeptides is used, the selectivity of adsorption is weak. In 
addition, adsorbability is weak in a narrow pH range which gives a 
relatively strong selectivity. Accordingly, the method is not applicable, 
unless phosphopeptides are prepared from an extremely diluted peptide 
mixture solution at the sacrifice of yield or production efficiency. 
Moreover, not only an ionic interaction takes part in the bond between an 
ordinary anionic exchange resin and peptide but hydrophobic bonds, etc. 
also strongly take part therein. Therefore, in order to sufficiently elute 
and recover a substance once adsorbed, it is necessary to use a solvent 
type eluent such as pyridine, or add a salt of a very high concentration, 
etc., which makes its application to practical preparation impossible. 
MEANS FOR SOLVING THE PROBLEMS 
The present invention relates to a method for separation and concentration 
of phosphopeptides which comprises treating a raw material comprising 
casein and/or a casein-based raw material with protease, adsorbing the 
resulting protein digestion products onto a crosslinked chitosan-formed 
substance and then desorbing the protein digestion products. The present 
invention also relates to the method wherein the adsorption is carried out 
in the pH range of 1.5 to 5.0. 
An object of the present invention is to solve the foregoing defects and to 
develop a large scale method which can efficiently isolate and recover 
phosphopeptides free of harmful or unnecessary minerals such as Ba, Ca, 
Fe, etc., without using expensive raw materials such as ethyl alcohol. 
Another object of the present invention is not to develop merely an 
industrial method suited for a large scale production but is to develop a 
large scale method in which devices already installed in the dairy 
industry or food industry can be used, without installing new apparatuses. 
Therefore, the present inventors have made various investigations on an 
economical preparation by concentrating and isolating phosphopeptides from 
protease digestion products of casein on an industrial scale, using 
devices conventionally installed in the dairy industry or food industry, 
without installing new apparatuses, without using any reagents unsuitable 
from a hygienic consideration. As a result, it has been found that the 
objects can be achieved by the use of a separating agent (hereafter 
crosslinked chitosan molding) prepared by molding chitosan (polysaccharide 
comprising 2-amino-2-deoxy-D-glucose as a main component), followed by a 
crosslinking treatment. The present invention has thus been accomplished. 
The chitosan-based crosslinked chitosan molding used in the present 
invention can be obtained by molding chitosan, then subjecting the molding 
to a crosslinking treatment, thereby imparting acid resistance to the 
molding. 
The chitosan molding may be of particles, layers, fibers, etc. but in view 
of surface area or operation upon use, the chitosan is preferably 
comprised of porous particles. 
The porous particles of chitosan may be obtained by the method disclosed in 
Japanese Patent Publication No. 16420/89. That is, the porous particles 
are obtained by dissolving lower molecular weight chitosan having an 
average molecular weight of 10,000 to 230,000 in an acidic solution such 
as an acetic acid solution, etc. to obtain a chitosan acidic solution of 2 
to 20%, adding dropwise the solution onto a basic solution such as a 
sodium hydroxide solution, etc. to regenerate and solidify, and then 
thoroughly washing with water. 
In this case, the particle diameter of the porous particles can be 
controlled by the nozzle diameter of a dropping nozzle of the chitosan 
acidic solution, a discharge pressure, and the like. 
The particle diameter of the chitosan porous particles is not particularly 
limited but is preferably 0.1 to 1.0 mm.phi. in the present invention, 
from aspects of operation and adsorption amount. 
The crosslinking agents used for the purpose of imparting acid resistance 
to the thus obtained chitosan molding may be those which are reactive with 
the amino group of chitosan and examples thereof include diisocyanates, 
dialdehydes, dicarboxylic acid derivatives, diepoxy compounds, etc. In 
view of ion exchange volume and reactivity, it is preferred to use diepoxy 
compounds shown by the following formulae. 
##STR1## 
(wherein n=1 to 10, m=3 to 10) 
In the formulae above, examples of [1] include ethylene glycol diglycidyl 
ether, diethylene glycol diglycidyl ether, triethylene glycol diglycidyl 
ether, etc.; and examples of [2] include trimethylene glycol diglycidyl 
ether, tetramethylene glycol diglycidyl ether, hexamethylene glycol 
diglycidyl ether, etc. 
When the crosslinking degree in this case is excessively high, a pore 
diameter becomes small, so that a diffusion rate of CPP in the particles 
decreases: when the crosslinking degree is too low, strength and acid 
resistance decrease. Therefore, it is preferred to treat with the 
crosslinking agent in a range of 5 to 30 mol % per chitosan residue. 
As casein which is used as a raw material in the present invention, a 
variety of acid casein, sodium caseinate, calcium caseinate, etc. are most 
preferred, but unpurified casein such as milk, skimmed milk, etc. may also 
be used. Concentrates or treated matters such as residues after 
ultrafiltration thereof, etc. may also be advantageously used. 
Furthermore, unpurified phosphopeptides or various phosphocaseinates, etc. 
may also be used. Thus, casein-containing or casein-based various raw 
materials may be widely used. 
In the present invention, such raw materials are treated with protease to 
produce protein digestion products. This step may be carried out as in the 
step for preparing CPP using trypsin as protease. For example, after acid 
casein is neutralized, or sodium caseinate is dissolved to form a solution 
of about 1 to about 10%, protease is added to the solution and the mixture 
is allowed to stand at pH of 8.0, a temperature of 30.degree. to 
45.degree. C. for 3 to 24 hours. From economical consideration, it is 
preferred to adjust pH and a ratio of soluble N/total N after completion 
of the enzyme reaction to 4.6 and 0.8 or more, respectively. 
In the present invention, protease is not limited only to purified trypsin 
as in the case of CPP but there may be used protease such as 
animal-derived enzyme such as pancreatin, pepsin, chymotrypsin, etc.; 
plant-derived enzyme such as papain, bromelain, ficin, etc.; 
micro-organism-derived enzyme such as mold, yeast, bacterial protease, 
etc. 
From a mixture solution of various peptides prepared by the digestion with 
protease of casein or material containing casein chiefly, acid peptides 
(chiefly phosphopeptides) can be adsorbed in a very high selectivity and 
adsorption rate onto a separating agent, i.e., the crosslinked chitosan 
molding as above-prepared. Upon elution of phosphopeptides an elution rate 
of about 80% or more can be easily achieved merely by varying pH. Where 
the elution rate should be as close as 100% at one, this can be achieved 
by adding a small amount of sodium chloride. 
While it is not sufficiently clarified yet that the crosslinked chitosan 
molding is excellent for phosphopeptides, it is assumed that positively 
charged amino groups are uniformly dispersed with appropriate intervals, 
hydrophilic property of the skeleton excluding the positively charged 
portions is strong, etc. would be effective. 
A concentration of the peptide solution mixture is not particularly limited 
but as a criterion of the concentration, the concentration obtained after 
the enzyme reaction is completed may be used as it is; or, if necessary, 
it is preferable to dilute the same to about 0.1 to about 5% of the solid 
content. 
In order to selectively adsorb phosphopeptides from the solution of casein 
and enzyme digestion products, it is most important to control pH. The pH 
value should be in the range of 1.5 to 5.0, more preferably, 2.5 to 4.5. 
At pH of greater than 5.0, adsorption capability is poor since both primary 
amino groups and secondary amino groups in the crosslinked chitosan 
molding have extremely small dissociation and their ionic interaction with 
phosphopeptides is weak. In addition, many peptides other than 
phosphopeptides in the casein enzyme digestion solution are negatively 
charged in such a high pH range, so that selectivity of adsorption becomes 
low. Conversely at pH below 1.5, charge state in most of the 
phosphopeptides is either extremely low or the same symbol as that of the 
crosslinked chitosan molding, so that no adsorption occurs. 
The adsorption temperature may be at room temperature. Even in an ordinary 
variation of temperature, any great change in adsorption rate is not 
observed. 
The adsorption of phosphopeptides begins at the moment when the enzyme 
digestion solution of casein is brought into contact with the crosslinked 
chitosan molding. However, in order to render the adsorption amount the 
maximum, it is preferred to take a reaction time of 10 to 30 minutes after 
pH adjustment. 
From the thus prepared phosphopeptide-adsorbed matter, phosphopeptides can 
be easily isolated and separated by conventional solid-liquid separation 
methods. For the solid-liquid separation, centrifugation, decantation, 
filtration, membrane filtration, etc. may be used but the separation is 
not limited only to these techniques. 
The crosslinked chitosan molding to which the acid peptide prepared as 
described above is adsorbed is treated to discharge the solution 
containing non-adsorbed peptides through a screen equipped in a reaction 
vessel and then, if necessary, wash off the crosslinked chitosan molding 
with water, and finally goes into the step of eluting phosphopeptides. 
That the desorption of most phosphopeptides can be effected simply by 
adjusting pH is characteristic of the use of the crosslinked chitosan 
molding. More specifically, the crosslinked chitosan molding after the 
non-adsorbed fraction is discharged is dispersed in water and its pH is 
adjusted with an acid or an alkali to 1.5 or less or 5 or more. 
Only by adjusting pH, about 10 to about 20% of the phosphopeptides adsorbed 
may remain in some occasion; in this case, the residual peptides increase 
by using repeatedly the crosslinked chitosan molding. In such a case, by 
adding approximately 0.2 to 1 mol/l of sodium chloride aqueous solution, 
the residual peptides can be eluted out and recovered. 
As an apparatus for adsorption and desorption (elution) of the 
phosphopeptides using the crosslinked chitosan molding, a stainless or 
synthetic polymer-made column packed with the crosslinked chitosan molding 
may be used but it is sufficient to use a simple system in which the 
enzyme digestion solution of casein is introduced into a tank or fermenter 
ordinarily used in the dairy industry and a definite amount of the 
crosslinked chitosan molding is incorporated therein. 
The eluted solution containing the phosphopeptides is concentrated and 
sterilized; the concentrate may be packed to provide for use as it is, or 
the concentrate may be spray dried to form powder. Where large quantities 
of salt are used during the course of elution, desalting may be performed 
by ion exchange or electric dialysis, etc. followed by concentration and 
drying. 
Next, preparation of the crosslinked chitosan molding used in the present 
invention and the isolation and concentration of the phosphopeptides are 
described in detail in the following examples. Test examples are also 
shown.

EXAMPLE 1 
After 1.8 kg of low molecular weight chitosan having a deacetylation degree 
of 82% and a mean molecular weight of 42,000 was dissolved in 27.9 kg of 
3% acetic acid solution, the resulting solution was added dropwise to a 
solution mixture of sodium hydroxide:ethanol:water=6.5:20:73.5 through a 
nozzle having a pore diameter of 0.15 mm.phi. to solidify and regenerate. 
Then, the product was washed until it became neutral. Thus, 30 l (wet 
state) of the chitosan molding having a mean particle diameter of 0.3 
mm.phi. was obtained. 
To 30 l (wet state) of the chitosan molding obtained were added 15 l of 
isopropyl alcohol and 300 g of ethylene glycol diglycidyl ether. After 
reacting at 70.degree. C. for 2 hours, the reacted molding was washed with 
water to give 30 l of the crosslinked chitosan molding having a specific 
surface area of 85.2 m.sup.2 /g and an ion exchange volume of 0.45 meq/ml. 
Separately, the pH of 100 kg of 5% casein solution prepared from acid 
casein was adjusted to 8 and using swine trypsin (Trypsin-6S, Novo Co., 
Ltd.), casein was decomposed until the nitrogen components insoluble at pH 
4.6 became 10% or less. The precipitates caused at pH of 4.6 were removed 
by centrifugation; about 3,960 g of peptide containing about 450 g of 
phosphopeptides was noted in the solution. The solution was transferred to 
a reaction tank equipped with a screen at the lower part thereof. After 30 
l (wet state) of the crosslinked chitosan molding prepared as described 
above was put in the solution, pH was adjusted to 3.5 with hydrochloric 
acid. The system was allowed to stand for 30 minutes; and then the liquid 
was discharged through the screen, whereby only the crosslinked chitosan 
molding remained. After the crosslinked chitosan molding was washed with 
50 kg of water, 100 kg of water was charged in the tank and pH was 
adjusted to 1.5 while gently stirring together with the crosslinked 
chitosan molding, followed by being allowed to stand for 20 minutes, 
thereby the acid peptides mainly composed of phosphopeptides being eluted 
out of the crosslinked chitosan molding. About 520 g of the solids mainly 
composed of the peptides were contained in the eluate; among them, the 
phosphopeptides were about 320 g. 
EXAMPLE 2 
To 30 l (wet state) of the chitosan molding having a mean particle diameter 
of 0.3 mm.phi. obtained in a manner similar to Example 1 were added 15 l 
of isopropyl alcohol and 325 g of trimethylene glycol diglycidyl ether. 
After reacting at 70.degree. C. for 2 hours, the reacted molding was 
washed with water to give 30 l of the crosslinked chitosan molding having 
a specific surface area of 85.5 m.sup.2 /g and an ion exchange volume of 
0.48 meq/ml. 
A solution of the digestion product of casein with trypsin was prepared 
under the same conditions as in Example 1 and the solution was transferred 
to the same reaction tank. After 30 l of the crosslinked chitosan molding 
prepared as described above was put in the solution, pH was adjusted to 
3.5 with hydrochloric acid. The system was allowed to stand for 30 
minutes; and then the liquid was discharged through the screen, whereby 
only the crosslinked chitosan molding remained. After the crosslinked 
chitosan molding was washed with 50 kg of water, 100 kg of water was 
charged in the tank and pH was adjusted to 7.3 while gently stirring 
together with the crosslinked chitosan molding, followed by being allowed 
to stand for an hour, thereby the acid peptides being eluted out of the 
crosslinked chitosan molding. About 590 g of the solids mainly composed of 
the peptides were contained in the eluate; among them, the phosphopeptides 
were about 360 g. 
TEST EXAMPLES 
Test examples are shown to compare the ability of adsorbing phosphopeptides 
between the crosslinked chitosan molding and known ion exchange resins and 
their elution behaviors. 
A solution of the digestion products of casein with trypsin containing 
0.45% of phosphopeptides was taken by 80 ml each in 5 beakers of 100 ml. 
To each beaker was charged 5 g each of the crosslinked chitosan molding 
prepared in Example 1 and four kinds of known ion exchange resins. After 
pH was adjusted to 3.5 with hydrochloric acid, the system was allowed to 
stand for 30 minutes. Using a glass funnel as a filter, the resin and the 
discharge liquid (A) containing non-adsorbed fraction were separated from 
each other. A volume of (A) and phosphopeptide content in (A) were 
determined. An adsorption rate of the phosphopeptides was calculated 
according to the following equation: 
##EQU1## 
Next, the crosslinked chitosan molding and ion exchange resins remained on 
the glass filter were restored to beakers of 100 ml, respectively. After 
50 ml of water was added into each beaker, pH was adjusted to 1.5 with 
hydrochloric acid. The system was kept for 30 minutes as it was, to elute 
the adsorbed phosphopeptides. Then, the resin and the eluate (B) were 
separated from each other using a glass funnel, and the phosphopeptide 
content in the eluate (B) was determined. An elution rate was calculated 
according to the following equation: 
##EQU2## 
The results are summarized in the following table. 
The results reveal that the crosslinked chitosan molding is extremely 
superior in efficiency. 
______________________________________ 
Adsorption 
Elution 
Skeleton 
Rate (%) Rate (%) 
______________________________________ 
I Crosslinked chitosan 
chitosan 88 82 
molding obtained in 
type 
Example 1 
II DIAION PA 308 styrene 62 4 
(made by Mitsubishi 
type 
Chemical Industry 
Co., Ltd.) 
III AMBERLITE IRA 35 
acryl 55 12 
(made by Organo Co.) 
type 
IV AMBERLITE IR 45 styrene 53 10 
(made by Organo Co.) 
type 
V DIAION WA 30 styrene 62 5 
(made by Mitsubishi 
type 
Chemical Industry 
Co., Ltd.) 
______________________________________ 
EFFECTS OF THE INVENTION 
According to the present invention, casein-derived phosphopeptides can be 
produced extremely economically on an industrial scale, without using any 
organic solvent such as ethanol, pyridine, etc., utilizing facilities 
already installed, in such a form free of calcium or iron that the 
phosphopeptides are expected to accelerate the absorption of calcium or 
iron in vivo. 
The thus obtained phosphopeptides can be used over wide areas including 
food materials, functional foodstuffs, nutrients, etc.