Comparison of hydrolysis time and precipitation pH in
the production of caseinophosphopeptides from a
casein by-product
E. Cruz-Huerta, M.J. García-Nebot, B. Miralles, I. Recio, L. Amigo*
Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), Spain
lourdes.amigo@csic.es
INTRODUCTION
The production of caseinophosphopeptides (CPPs) from a casein-derived by-product generated during the manufacture of an antihypertensive hydrolysate was attempted. CPPs are
phosphorylated bioactive peptides that can be released from casein by enzymatic digestion. Due to the phosphorylated serines, these peptides are relatively resistant to be further
hydrolysed and it has been proposed that they could prevent the precipitation of metal ions at alkaline pH in the distal small intestine. In order to isolate CPPs from the casein fraction
of milk, an enzymatic hydrolysis process is often followed by selective precipitation with calcium chloride and ethanol using pHs in the range 3.5 to 8.5. It has been reported that at pH
3.5 only peptides containing the phosphorylated cluster sequence SerP-SerP-SerP-Glu-Glu selectively precipitate from a tryptic digest of casein. However, at higher pH an increase of
the recoveries of the di- and monophosphorylated peptides was observed.
OBJECTIVE
The objective was to investigate if the hydrolysis time and pH used for the selective precipitation could determine the characteristics of the products recovered using tandem mass
spectrometry for peptide sequencing. The resulting CPPs were compared to those released after gastrointestinal simulated digestion previously identified.
αs1-CN 120 min
MATERIALS AND METHODS
pH 4
pH 6
pH 8
Peak area (Arbitrary Units)
1.4E+07
Milk casein
1.2E+07
1.0E+07
8.0E+06
6.0E+06
4.0E+06
2.0E+06
0.0E+00
f(43-52)2P
f(43-55)2P
f(43-56)2P
f(43-59)2P
Figure 1. The diphosphorylated αs1-CN fragments 43-52, 43-55, 43-56 and 43-59 showed
higher relative amounts in the pH 8.0 precipitates than the pH 4.0 and 6.0 precipitates.
Casein by-product
αs2-CN (56-68)3P
Peak area (Arbitrary Units)
Hydrolysis with trypsin
2% (w/w) 50°C
Hydrolysis time:
30,60,120 min
pH
pH 4
pH 6
pH 8
2.50E+07
2.00E+07
1.50E+07
1.00E+07
5.00E+06
0.00E+00
30 min
selective precipitation:
4.0, 6.0 and 8.0
60 min
120 min
Figure 2. Peptide αs2-CN f(56-68)3P presented a higher relative recovery at the pH 6.0
precipitate at all the studied times. This peptide was formed from 30 min and its relative
amount decreased at 120 min of tryptic hydrolysis.
Identification and semi
quantification of CPPs by
β-CN (pH 8)
RP-HPLC-MS/MS
Peak area (Arbitrary Units)
1.20E+07
RESULTS
A total of 44 CPPs were identified, of which, 13 corresponded to αs1-CN,
11 to αs2-CN, 18 to β-CN, and two to -CN. It must be pointed out that 13
of the identified CPPs presented the cluster sequence (SpSpSpEE). They
corresponded to αs1-CN f(61-79), αs2-CN f(1-20), αs2-CN f(46-70) and βCN f(1-25) or fragments thereof. All of the peptides were found in every
hydrolysis product, with the exception of αs1-CN (61-79)5P, which was
only found in the precipitates at pH 8.0. The common CPPs identified in
the trypsin hydrolysates and simulated digests represented 73% of the
total sequences.
6.00E+08
f(12-25)
1.00E+07
5.00E+08
f(17-25)
f(7-25)
8.00E+06
4.00E+08
f(1-25)
f(2-25)
6.00E+06
3.00E+08
4.00E+06
2.00E+08
2.00E+06
1.00E+08
0.00E+00
0.00E+00
0
20
40
60
80
100
120
140
Time (min)
Figure 3. The decrease of β-CN f(1-25)4P showed some susceptibility of the parent
peptide to the trypsin activity. However, the release and no further degradation of β-CN f(725)4P and f(12-25)4P permits to keep the biological activity.
CONCLUSIONS
The precipitation at pH 6 and 8 permitted to recover a valuable CPPs fraction and use this casein-derived by-product as source of CPPs. Identification of CPPs in the samples of CNderived by-product after being subjected to simulated gastrointestinal digestion showed that this process generates CPP sequences very similar to those produced by tryptic
hydrolysis. These results suggest that this CN-derived by-product can be used as a source for CPPs after its passage through the gastrointestinal tract opening the door for its use as
functional ingredient to improve mineral bioavailability.
ACKNOWLEDGEMENTS
This work has received financial support from projects AGL2011-24643, FEDER-INNTERCONECTA-GALICIA (ENVELLEFUN), and FP7-SME-2012-315349 (FOFIND). The authors are participants in the
FA1005 COST Action INFOGEST on food digestion. E. C-H. thanks, scholarship PROMEP/103.5/13/6408 for the support for PhD studies abroad