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AIR2-CT94-1560
Health and Quality Enhancing Components from Milk Proteins for Food and Pharmaceutical Applications |
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Proposal No: | AIR2-CT94-1560 |
| Date Prepared: | April 1998 | |
| Source: | Progress and Final Reports |
Objectives
The overall aim of this project was the development
of milk protein hydrolysates with reduced allergenicity and enhanced content of
phospho-, bioactive peptides. This was to be achieved through the following
objectives:
In the course of this work it was proposed to optimise industrial-scale processes for the production of hydrolysates having these properties, through collaborative work programmes in the following areas:
In the final year participants had to:
Technical summary
Work on the development and characterisation
of caseinophosphopeptides (CPPs) concentrated on their production using three
specific enzyme systems: PTN 3.0S, Pancreatin and Bioprotease N100. CPP yield
ranged from 9.2 - 17.1 % (w/w) when using these enzymes in the hydrolysis of
sodium caseinate. The calcium solubilising abilities of these CPPs ranged from
19.1 to 25.0 mg calcium solubilised per mg CPP. Simulated in vitro digestion
experiments with these CPPs indicated that they were resistant to further
degradation by gastrointestinal proteinases. Functional characterisation of the
CPPs show that the CPPs generally had good solubility over a range of pH values.
Fortification of model food systems i.e. mayonnaise and orange juice showed that
CPP addition to these products resulted in acceptable taste. Detailed
characterisation of the CPPs produced with the Bioprotease preparation resulted
in the identification of a CPP not previously described (beta-casein f(33-44)
1P).
A procedure has been developed for the production of casein hydrolysates which are free of intact whey proteins. The protocol involves digestion of casein with a bacterial enzyme which under the experimental conditions does not hydrolyse whey proteins. Subsequent ultrafiltration of the hydrolysate through 10 or 30 kDa membranes has the effect of separating the intact whey proteins from the hydrolysed casein. Analysis of the IgE response in sera derived from 20 patients allergic to bovine beta-casein resulted in the identification of two highly immunoreactive regions which are common to bovine and human beta-casein. The transfer of a laboratory-scale process for the production of reduced allergenicity casein hydrolysates to the pilot-plant scale met with several problems. Some of these problems have been addressed, for example electrodialysis has been incorporated into the production process as a means of lowering the salt levels within the casein hydrolysates prior to the spray-drying step.
Angiotensin-I-converting enzyme(ACE) inhibitory activity has been demonstrated for the first time in a wide range of dairy products especially ripened cheese. Tissue culture analyses of peptides previously shown to have ACE inhibitory activity showed that these peptides give different responses depending on the cell types (PBL and HL-60) under study.
The functional properties of purified beta-lactoglobulin, purified beta-casein and sodium caseinate hydrolysates generated with a range of different enzyme systems were studied. In general it was concluded that the functional properties i.e. the emulsification and foam forming and stabilising properties of low degree of hydrolysis beta-lg hydrolysates differed little from those of the intact protein. Distinct differences in the functional properties of fractionated plasmin digests of beta-casein were observed. It was concluded that the N-terminal hydrophilic region of beta-casein was involved in emulsion stabilisation. Bacillus proteinase generated digests of sodium caseinate with a low degree of hydrolysis were shown under certain conditions to have enhanced emulsion activity, increased foam expansion and reduced foam drainage characteristics.
Methods
In general hydrolysates were generated from casein, whey
protein concentrate and individually isolated milk proteins using a range of
individually isolated enzymes and/or commercially available crude enzyme
preparations. Hydrolysate fractions and individual peptides were obtained by a
combination of ultrafiltration, selective enrichment, solid phase extraction and
analytical/semipreparative chromatographic techniques. Physicochemical
characterisation of hydrolysates was performed using high performance liquid
chromatography, mass spectroscopy (electronspray ionisation and fast atom
bombardment) and nuclear magnetic resonance analysis. Hydrolysate functionality
was assessed using an electrical conductivity approach to determine foam
capacity and stability. The average particle size and the particle size
distribution of emulsions prepared with hydrolysates and fractions therefrom was
measured using a Malvern Mastersizer. Sensory analysis was performed using a
taste panel trained to detect bitterness. Calcium binding/solubilisation of
phosphopeptide fractions was carried out using a specific calcium colourimetric
reagent and also by atomic absorption spectroscopy. Calcium binding constants
were measured using capillary electrophoresis. ACE inhibitory activity was
determined using the standard photometric assay. Residual antigenicities in the
casein hydrolysates were determined using IgE containing serum obtained from
human donors displaying clinical symptoms of allergy to cows milk protein.
Native casein was dephosphorylated using immobilised potato acid phosphatase.
Cells for tissue culture experiments were either obtained from public culture
collections, or were isolated from human blood samples. The effect of peptides
on cell proliferation was quantified by determining the uptake of
5-bromo-2'-deoxy-uridine in DNA using an ELISA detection assay. Protein
biosynthesis was quantified radiochemically by determining the uptake of
tritiated leucine by cells. Peptide induced cell death or apotosis was measured
using an antibody system to determine cytoplasmic histone-associated DNA
fragments.
Results
During the second year of this project the participants:
Achievements - year 2.
During the second year casein
hydrolysates were generated at laboratory scale which display significant
reductions in residual antigenicities as determined using human IgE antibodies
to casein,**-lactoglobulin, bovine serum albumin and bovine IgG. These
hydrolysates, however, contain relatively high levels of residual antigenicity
to a-lactalbumin and work is in progress to find means of reducing the a-
lactalbumin associated residual antigenicity. Several difficulties were
encountered during pilot scale trials to produce these hydrolysates. These
difficulties related to - problems in spray drying the final hydrolysate due to
the presence of high salt levels ,Arising from the requirement to add alkali
during the hydrolysis process in order to maintain constant pH. These
difficulties have been overcome by first subjecting the 7 hydrolysate to
electrodialysis to remove excessive salt. Following electrodialysis, the
hydrolysate was easily spray dried to give kilogram quantities of a reduced
antigenicity casein hydrolysate.
Work has progressed in determining the antigenicity of casein derived from different animal species. Differences were observed in the IgE responses to ruminant and non- ruminant caseins. Immunoreactivity studies on a range of beta-casein derived peptides show that the IgE response was variable and heterogeneous and that the , beta-casein allergenic epitopes were widely spread along the entire sequence of the protein molecule. A sensitive assay has been developed for the ABBOS peptide, a BSA derived peptide, implicated in the development of diabetes mellitus in susceptible individuals.
A range of commercially available enzymes have been screened for their ability to produce caseinophosphopeptides(CPPs) from sodium caseinate. Differences in CPP yield and in calcium solubilising abilities have been observed when using different enzymes for hydrolysis. Kilogram quantities of CPPs have been produced in the course of pilot plant CPP production trials. A modified laboratory procedure has been developed to isolate individual CPPs from a tryptic digest of sodium caseinate.
Novel ACE inhibitory activity has been detected in whey protein hydrolysates. A tryptic ACE inhibitory peptide derived from ,beta-lactoglobulin has been shown to be resistant to further degradation by pepsin and chymotrypsin. As a consequence this peptide may find application in the prevention of hypertension following oral ingestion. Very potent ACE inhibitory peptides have also been isolated from tryptic digests of sodium caseinate. Detailed structural characterisations have been performed on casein glycomacropeptide (CMP) and peptides derived therefrom. CMP peptides have potential application as antithrombic agents. Studies on the surface properties of a range of beta-casein peptide fractions show difference in emulsification and surface activities. Studies are ongoing on the kinetics and thermal stability of immobilised papain. Preliminary results indicate differences in the reversed phase HPLC profiles of beta-casein hydrolysates obtained by soluble as opposed to immobilised papain. Market assessments show expanding consumer awareness of the relationship between diet and health. The market for bioactive milk proteins/peptides is estimated to be around $80m in the year 2000.
Results year 3
The method for producing casein hydrolysate free
of intact whey proteins developed involved the digestion of total casein (which
can contain minor trace of whey proteins) with a purified cell-envelope
proteinase (CEP) isolated from a Lactococcus bacterium. The CEP from
Lactococcus lactis subsp. cremoris W92 is unable to hydrolyse
intact whey proteins i.e. BSA, alpha La, beta-lg and IgG. Ultrafiltration of
total casein hydrolysed with the CEP through 10 or 30 kDa molecular mass cut-off
membranes results in the retention of BSA. In this way casein hydrolysates free
of the BSA-derived ABBOS (antibody binding sites can be produced).
The IgE response in sera derived from 55 patients allergic to bovine beta-casein, was found to, in general, be lower for native as opposed to dephosphorylated beta-casein. It was shown using native and dephosphorylated beta-casein f(1-28), that in some individuals by indirect competitive ELISA, that dephosphorylated beta-casein could not inhibit IgE binding to beta-casein f(1-28). The specificity of the sera from 20 patients allergic to bovine beta-casein toward human beta-casein was tested by direct and indirect competitive ELISA's. The results indicate some cross-reactivity between human and bovine caseins. Furthermore, indirect competitive ELISA-based IgE inhibition tests to immobilised human beta-casein in the presence of various bovine casein peptides show two highly immunoreactive regions presumably responsible for the cross-reactivity between human and bovine beta-caseins. These peptides correspond to beta-casein f(l-52) and f(l06-139).
Laboratory-scale experiments on the development of reduced antigenicity casein hydrolysates concentrated on the reduction of residual antigenicity to alpha-la in casein hydrolysates. A system employing four different enzyme digestion steps can lead to casein hydrolysates having very low levels of residual antigenicity to whole casein, BSA, beta-lg and IgG with significant reductions in the residual antigenicity to alpha-la. The appearance/disappearance of residual antigenicity to alpha-la in the casein hydrolysates was time dependent. A lack of reproducibility in this time dependence was also observed.
Laboratory-scale studies on casein hydrolysates at 50 ml and 5l scale to produce caseinophosophopeptide (CPP) enriched preparations were carried out using 3 specific enzyme preparations. The results show that CPP yield following calcium/ethanol and/or Ca2+ aggregation followed by ultrafiltration ranged from 9.2 17.1%. CPP yield was dependent on the CPP enrichment step used being higher with the Ca2+ aggregation and UF procedures. The calcium solubilizing abilities of these preparations ranged from 19.1 - 25.0 mg Ca2+ solubilized per mg CPP. Simulated in vivo digestion of the CPP preparations indicated a general resistance to further degradation by gastrointestinal proteinases as deterrnined following gel permeation and reverse-phase HPLC analysis. The phosphorus:serine ratios as deterrnined for the laboratory-scale CPP preparations were high (i.e. > 1.3) indicating that it was possible to produce good quality CPP's at laboratory-scale.. Large-scale hydrolysis reactions (5001) were carried out in using 3 selected commercially available food-grade enzymes. The P:Ser ratios for these samples- was < 1.0, indicative of poor quality CPPs. CPP yield ranged from 12.0 - 13.2%, values somewhat lower than those obtained at lab-scale. Furthermore, the Ca2+ solubilising abilities of the large-scale CPP's were lower than those produced at lab-scale. The protein solubilities of the large-scale CPP's were good over a range of pH values. Detailed Characterisation of the individual CPP's present in the enriched CPP produced with Bioprotease resulted in the identification of a new CPP [beta-casein f(33 - 44) lP], that has not previously been described in the literature.
Tissue culture experiments with bioactive peptides derived from bovine alpha-lactalbumin and beta-lactoglobulin showed different effects in the in vitro modulation of cell proliferation and apoptosis. Tyr-Gly had a stimulatory effect on proliferation of PBL and HL-60 cells; protein biosynthesis was also enhanced. PBL showed a proliferative response to Ile-Arg and Arg-Leu over the whole concentration range; no significant effect was obtained with HL-60 cells. His-Ile-Arg suppressed the proliferation of PBL, but not of HL-60. Apoptosis of PBL was induced by His-Ile-Arg over the whole concentration range. Ala-Leu-Pro-Met-His-Ile-Arg and Ala-Leu-Pro-Metamide showed no significant effect on both cell types.
The functional (emulsifing, foam forming and stabilising) properties of beta-lg hydrolysates generated with plasmin, trypsin and S. aureus V8 proteinases were determined pH 4.0 and 6.7. In general it was concluded that hydrolysates at DH % values of 1, 2 and 4% did not result in significantly different functional properties compared to unhydrolysed material. A plasmin hydrolysate of beta-casein was separated into eight different fractions. The peptide components in these fractions were characterised by mass spectrometry following preparative RP HPLC. The peptides were divided into 5 different groups e.g. hydrophobic, arnphiphilic, etc. Distinct differences in the emulsion and foaming properties of the different peptide fractions were observed during analysis performed at pH 4.0 and pH 6.7. One fraction designated RET-l was further fractionated by ion-exchange. chromatography into 3 distinct peaks. The foam and emulsion forming properties of peaks 2 and 3 were found to be similar. However, peak 2 had poor emulsion stabilising properties whereas peak 3 had emulsion stabilizing properties as good as RET-I. Differences in the emulsion stabilizing abilities of peaks 2 and 3 were related to the presence of the hydrophilic N-terminal region of ,beta-casein in peak 3. Sodium caseinate hydrolysates generated with a Bacillus proteinase were shown at low DH% values to have increased emulsion activity at low pH in comparison to unhydrolysed sodium caseinate. Increased foam expansions and decreased foam drainage was observed for the hydrolysates at high pH values At a DH % value of 0.5 %, the Bacillus proteinase hydrolysate was significantly less bitter than a tryptic hydrolysate of sodium caseinate at a similar DH %.
It was demonstrated that whey protein hydrolysates produced with a range of endoproteinases could inhibit ACE. In addition it was demonstrated that ultrafiltration could be used to enrich for ACE inhibitory peptides in whey protein hydrolysates. It was also shown for the first time that ACE inhibitory activities can be found in a range of dairy products, especially ripened cheese. Papain and cathepsin H and D were immobilised onto agarose solid supports. The activity of papain was substantially reduced whereas the kinetics of cathepsin D appeared to be unaltered following immobilisation.
Discussion
The application of a specific enzymatic activity in
conjunction with ultrafiltration to remove whey proteins from caseins represents
a significant development for the production of casein hydrolysate-free whey
proteins and/or the production whey protein-free casein hydrolysates. It should
be of interest to up-scale this process to pilot plant scale.
The identification of the major potential allergenic sites from studies on human and bovine beta-casein represents a significant advance. Knowledge of these sites allows the potential to develop a targeted, as opposed to an empirical, approach to the production of reduced/hypoallergenic casein hydrolysates for human consumption.
While a production protocol has been developed to obtain casein hydrolysates having significant reductions in their allergenic potential much work is still required to reproducibility produce these hydrolysates. However, it has been demonstrated, using an empirical approach, that it is possible to find a specific combination of proteolytic enzymes to produce such hydrolysates.
Significant advances have been made in the production and characterisation of novel caseinophosphopeptides (CPPs) This study would appear for the first time to have performed a detailed characterisation on the contribution of gastrointestinal and bacterial proteinases to both the laboratory and pilot-scale development of CPPs. While food functionality results are promising, further work (to be addressed in an upcoming FAIR project) is required to optimise the production and application of CPPs.
It has now been demonstrated that ACE inhibitory activities are present in a range of dairy products including whey protein hydrolysates and ripened cheese. This expands the potential of exploiting milk proteins as nutraceutical/functional food ingredients. The use of tissue culture techniques to study the effects of specific milk-protein derived peptide sequences on cell proliferation, apoptosis, etc., represents a major advance in the characterisation of bioactive peptides.
Bitterness is a major negative attribute associated with hydrolysed casein products. Development of hydrolysates with improved functionality and lower bitterness helps expand the applicability of hydrolysed casein products. Detailed studies on the functionality of specific ,beta-casein-derived peptides allows the establishment of structure - function relationships and long term will expand the applicability of milk protein hydrolysates as functional (i.e. foaming and emulsifying) food ingredients.
© Copyright 2006 Policy Statements
Updated
by CPL Press:
03/07/2007
- biomatnet@biomatnet.org
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