1. 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. OBJECTIVEOBJECTIVE Comparison of hydrolysis time and precipitation pH in the production of caseinophosphopeptides from a casein by-product 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 CN- derived 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. 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 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 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. CONCLUSIONS RESULTS MATERIALS AND METHODS INTRODUCTIONINTRODUCTION A total of 44 CPPs were identified, of which, 13 corresponded to α s1 -CN, 11 to α s2 -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 α s1 -CN f(61-79), α s2 -CN f(1-20), α s2 -CN f(46-70) and β- CN f(1-25) or fragments thereof. All of the peptides were found in every hydrolysis product, with the exception of α s1 -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. Milk casein pH selective precipitation: 4.0, 6.0 and 8.0 Casein by-product Hydrolysis with trypsin 2% (w/w) 50°C Hydrolysis time: 30,60,120 min Identification and semi quantification of CPPs by RP-HPLC-MS/MS 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(7- 25)4P and f(12-25)4P permits to keep the biological activity. Figure 2. Peptide α s2 -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. Figure 1. The diphosphorylated α s1 -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.