Deliverables
The assessment of any reactive potential of CPPs, i.e. incidence on the immune response, has been studied in human volunteers during a six week feeding trial performed at IFR-N. A two-site immunometric (sandwich) ELISA test for anti CPPs specific IgE, IgG1, IgG2, IgG3, and IgG4 has been developed. It was applied to specific determination of IgE and IgG sub-classes in blood samples collected during the feeding trial.
Main conclusions were that:
1) No IgE response was induced.
2) A great heterogeneity of the "basal" human IgG response to CPPs in both intensity and specificity was observed before the beginning of the feeding trial (week 0)
3) No evolution of the IgG (e.g. IgG1 , IgG2, IgG3 and IgG4) response neither in intensity nor in specificity was detected all along the feeding trial. Therefore there is no observed immunological effect of the CPPs, at least on the antibody response, in humans during a long term feeding trial.
However no conclusion can be drawn in terms of risk assessment of any allergic potential of CPPs (as well as any milk derived product) to possibly trigger an allergic reaction in allergic consumers, already sensitised to milk proteins, who would ingest CPPs.
The practical implications arising from physical stability and sensory standpoints, if CPPs are added to consumer food products for fortification purposes, are very important from ultimate consumer and food manufacturer perspectives. Incorporation of CPPs into milk protein based model systems; ice cream and yoghurt had both neutral and negative effects. In the case of physical stability, the CPPs didn't cause problems when used in ice cream. However, when used in the model milk protein systems and in yoghurt the results showed that CPP addition had a very clear-cut negative effect on heat stability.
On the sensory front, the negative effect on flavour, due to addition of CPPs to both ice cream and yoghurt, were very obvious. It should be possible to overcome the flavour problems through the use of partial demineralisation of the CPPs or the use of masking ingredients. With regard to the poor heat stability findings, again demineralisation may help, or the adoption of modified consumer product manufacturing protocols, including CPP addition after heat treatment or the use of stabilisers, could prove useful.
During the production of CPPs, co-product streams depleted in CPPs are generated. These co-products may represent between 85 -90% of the starting substrate. Therefore, suitable outlets for these co-products are necessary in order to maintain the cost effectiveness of the whole CPP production process. The physicochemical (electrophoretic and chromatographic) and functional (solubility, emulsifying and foaming) properties of the CPP deplete co-products were therefore characterised with a view to identifying potential added value outlets.
Cytochemical studies aimed at the characterisation of dose-dependency concerning the cell activity and possible cytotoxic response of human cell culture model systems (PBL, HL-60, Caco-2, PML) to caseinophosphopeptide (CPP) preparations.
The results obtained with different cytochemical assays showed no apoptotic, antiproliferative or general cytotoxic effect of enriched CPP preparations made from casein hydrolysates when applied in physiologically relevant concentrations. Based on the absence of adverse effects, CPP preparations can be rated harmless in regard to cellular level. Besides no trophic properties were identified, i.e. cell development and metabolism under CPP treatment was comparable to untreated control cells.
PBL directly exposed to CPP preparations showed increase in IgG production pointing to a possible immuno-modulating activity of the phosphorylated peptides.
The study findings suggest that, although only a minority of consumers understand new terms like functional foods, there is considerable public awareness, mixed with some scepticism and confusion, that foods and diet can have both negative and positive impacts on health. Further evidence of this awareness can clearly be seen on the ground at food manufacturer and retailer levels in terms of growing consumer demand for and interest in health/lifestyle enhancing foods.
The findings also suggest that, in terms of the consumer message, consumers generally react more positively to soft, more lifestyle oriented health messages than to hard science/health messages with disease connotations, and that emotions have a greater effect on food choice than logic. These and other findings should provide better insights into what goes on in the consumer's mind when s/he is faced with making a functional food/nutraceutical product purchase decision and could prove useful and crucial to gaining consumer acceptance for functional foods, like CPP fortified foods, in the market place.
The findings also provide insights into what influences health professionals when providing health/nutrition related advice to patients (consumers) and could also prove to be very useful in the context of understanding consumer behaviour.
A unique competitive Enzyme Immuno Assay (EIA) with a broad specificity and a high sensitivity has been validated for detection and semi-quantification of "the whole CPPs" present in preparations obtained using different conditions of enzymatic hydrolysis.
Polyclonal antibodies have been raised in rabbits using a mixture of purified and characterised phosphorylated peptidic fragments prepared by tryptic hydrolysis of aS1-casein, aS2-casein and b-casein then cross linked together with keyhole limpet hemocyanin (KLH) as immunogen.
A phosphorylated peptide has been chemically synthesized. It contains a short consensus sequence of phosphorylated seryl residues, in order to cross react with antibodies specifically raised against tryptic CPPs and thus be used as tracer/standard to allow "non specific" detection of all CPPs sharing this same sequence. It has been labelled with Acetyl cholinesterase (AChE) and used as a tracer.
This combination has proved to permit a sensitive and reliable detection and semi quantification of the global CPPs content in four pilot preparations.
It has also been applied successfully to the detection of CPPs in ileostomy fluids obtained from humans fed CPPs or milk products.
It was shown for the first time that CPPs promote the uptake of extra-cellular calcium by human intestinal HT-29 cells, a tumoral cell line that undergoes enterocytical differentiation in culture. The molecular features underlying this biological effect were explored, using the following CPPs: alphas1-CN(59-79)5P from alphas1-casein, beta-CN(1-25)4P from beta-casein, both carrying the characteristic calcium binding "acidic motif" Ser(p)-Ser(p)-Ser(p)-Glu-Glu, and several properly designed derivatives of beta-CN(1-25)4P that were chemically synthesized.
CPPs were administered to differentiated HT-29 cells in 2 mM extracellular calcium, and cytosolic calcium changes were measured by the fura-2 method. The main results were as follows: beta-CN(1-25)4P is more active than alphas1-CN(59-79)5P; the dephosphorylated form of beta-CN(1-25)4P is poorly active; the peptide corresponding to the "acidic motif" is inactive; the peptide lacking the segment located C-terminally to the "acidic motif" maintains activity, whereas those without the N-terminally located segment or lacking the "acidic motif" loose activity; the peptide where the tetrapeptide 1-4 is exchanged with the tetrapeptide 8-11 is inactive, like that missing the first five amino acids.
Therefore, the calcium rise effect of beta-CN(1-25)4P appears to be due to the overall conformation of the molecule, with a primary role played by (a) the phosphorylation of the "acidic motif", (b) the loop-like structure of the first four amino acids, and (c) the beta-turn structures exhibited by the amino acid sequences 8-11 and 17-21, the latter one constituting the "acidic motif". Remarkably, the "acidic motif" per se is inactive, but its presence in the CPP is necessary for the calcium effect. These data point at a molecular attitude of CPPs to affect calcium transporters or sensors at the plasma membrane, possibly on intestinal cells, resulting in facilitation of calcium absorption. This property is consistent with a potential role of CPPs as functional food or food ingredient.
CPPs were generated from sodium caseinate by enzymatic hydrolysis using commercially available enzyme preparations. CPPs were enriched from these hydrolysates using either calcium aggregation followed by ethanol precipitation or calcium aggregation followed by ultrafiltration schemes. The enriched hydrolysates were then spray or freeze dried. The preparation processes were commercially significant.
The resultant powders were analysed for nitrogen and phosphate composition as well as calcium-binding and solubilising abilities. Samples of the CPPs were distributed to the partners for immunological and cytochemical analysis, use in functional studies and for use in in-vitro and human feeding experiments. The CPP-depleted hydrolysates were dried and distributed to the University of Limerick for application studies.
CPP-enriched hydrolysates were extensively characterised with respect to CPP composition by RP-HPLC and gel-permeation HPLC and in vitro calcium-binding and solubilising activities.
CPPs produced at pilot-scale were able to bind 1.95- 2.65mMol Ca per pg of peptide.
These hydrolysates were also characterised for their resistance to both simulated digestion with pepsin and gastrointestinal proteases and in vivo digestion in ileostomy patients. Results of analyses indicated that the CPPs were subjected to extensive degradation in the presence of such proteases with concomitant effects on calcium-binding abilities (dropping to 0.80mMol Ca per mg peptide).
Spray-drying conditions were optimised to prevent heat-induced dephosphorylation of the peptide preparations. Kg quantities of the preparations were produced and distributed to the partners for further characterisation.
Collaborative studies with the other partners have shown that these preparations were safe for human consumption.
A randomised crossover trial was performed to determine calcium absorption from a calcium lactate drink with or without (control) the addition of two CPP-enriched preparations using a 3:1 ratio of CPP to calcium in fifteen healthy adults. The dual-label calcium stable isotope technique was used to determine fractional absorption based on the measurement of calcium isotopic enrichment in urine by inductively coupled plasma mass spectrometry. In terms of fractional absorption the only significant difference was between the control drink and one of the two CPP enriched drinks (p = 0.015). The observed difference is unlikely to be of any nutritional significance when taking into account the variability surrounding absorption values and the small effect that the difference in dose might have had on these results. The experimental design did not allow to test various assumptions this study was based on and therefore, a clear answer to the effect of CPPs on the efficiency of calcium absorption requires further investigation.
Caseinophosphopeptides (CPPs) were detected in ileostomy fluid, collected at 2-hour intervals for 10-hour post milk and CPP ingestion, from human volunteers (ileostomists). The levels of CPPs present in ileostomy fluids obtained from milk fed volunteers was significantly higher than that in volunteers fed with selected CPP preparations.
The findings are based on HPLC analysis in combination with peptide-bound phosphorous determination, thin layer electrophoresis, amino acid analysis along with ELISA studies using polyclonal antibodies raised against a set of CPPs to detect immunoreactive CPPs in ileostomy fluid. These procedures allowed the detection of nanomolar concentrations of CPPs. CPPs, which can be released during intestinal digestion, may function as bioactive constituents and carriers for different minerals, especially calcium.
For their function as carriers CPPs must, at least partially, resist enzymatic digestion during intestinal passage. The present studies demonstrate, for the first time, that CPPs can survive the prolonged intestinal passage to the distal small intestine (ileum) in vivo that is a prerequisite for their function as bioactive substances.
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