- Email: [email protected]
Emulsion and foam properties of plasmin derived β-casein peptides
International Dairy Journal 9 (1999) 347}351
Emulsion and foam properties of plasmin derived b-casein peptides P.W.J.R. Caessens , S. Visser, H. Gruppen , G.A. van Aken, A.G.J. Voragen * Department of Food Science, Wageningen Agricultural University, P.O. Box 8129, 6700 EV, Wageningen, The Netherlands Department of Biophysical Chemistry, Netherlands Institute for Dairy Research (NIZO), P.O. Box 20, 6710 BA, Ede, The Netherlands
Abstract In the present study the in#uence of plasmin hydrolysis of b-casein (bCN) on the foam and emulsion properties was tested. The hydrophilic, amphipathic and hydrophobic fractions produced by plasmin hydrolysis of bCN and fractionation of the hydrolysate, show clear di!erences in emulsion, foam and surface-active properties. The hydrophilic peptide possessed poor functional properties. The hydrophobic peptides from bCN showed interesting foam properties, especially at acidic pH. The amphipathic peptides exhibited improved emulsion-forming properties, compared to intact bCN. It seemed that the presence of the hydrophilic N-terminus (i.e. fragment 1}28) in these amphipathic peptides was important for their stabilising properties. 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Milk proteins are well known for their surface-active behaviour and their ability to form and stabilise foam and emulsions. Chemical, physical, enzymatic, and genetic modi"cation can be applied to alter the functionality of proteins (Phillips, Whitehead & Kinsella, 1994). Enzymatic modi"cations have the advantage of mild reaction conditions (Arai & Fujimaki, 1991; Phillips et al., 1994), and can be used to enhance some functionalities of food proteins (Panyam & Kilara, 1996). Plasmin is a proteolytic enzyme with a preference for Lys}X and Arg}X bonds. In fresh milk it converts b-casein (bCN) into the c-caseins, and to the complementary parts called proteose peptones (Andrews, 1978a,b; Eigel et al., 1984). Wilson, Mulvihill, Donnelly and Gill (1989) used plasmin to modify bCN to yield c-caseins and proteose peptones. The surface pressure at the air/water (A/W) interface induced by these hydrolysis products appeared to be di!erent from that of the intact protein. Therefore, it is likely that the foaming and emulsifying properties of bCN will be altered by plasmin hydrolysis as well. The objective of the present study was
* Corresponding author. Tel.: #31-317-48-32-09; fax#31-317-4848-93. E-mail address: [email protected] (A.G.J. Voragen)
to produce, by means of plasmin hydrolysis, peptides from bCN with altered foam and emulsion (and surfaceactive) properties, with the aim of identifying structurefunction relationships.
2. Material and methods Bovine bCN (90% w/w bCN based on dry weight) was purchased from Eurial (Rennes, France). Bovine plasmin (EC 3.4.21.7) was obtained from Sigma (St. Louis, USA; product number P-7911). Unless stated otherwise, all other chemicals were of analytical grade, and were purchased from Merck (Darmstadt, Germany) or BDH (Poole, UK). Plasmin hydrolysis of bCN and fractionation of the hydrolysate have been described elsewhere (Caessens, Gruppen, Visser, van Aken & Voragen, 1997). An outline of the hydrolysis and fractionation conditions applied is shown in Fig. 1. All peptide fractions were lyophilised and stored at 43C before further analysis. Ion-exchange chromatography (IEC) was performed on the AG KTA-explorer, controlled by a UNICORNcontrol system (Pharmacia Biotech, Uppsala, Sweden) using a SourceQ column (280 ml bed volume; Pharmacia). The gradient formed with solvent A (20 mM Tris/ HCl, pH 8) and solvent B (20 mM Tris/HCl, 1 M NaCl, pH 8) is given in the IEC-"gure shown. Except where
0958-6946/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 0 8 6 - 2
348
P.W.J.R. Caessens et al. / International Dairy Journal 9 (1999) 347}351
Fig. 1. Outline of the bCN hydrolysis and fractionation of the hydrolysate. Table 1 MS results for the RP-HPLC collected peaks of bCN/plasmin hydrolysates, categorized in RP-HPLC groups I}V (see Fig. 2) RP-HPLC groups
Fig. 2. RP-HPLC chromatograms of the bCN hydrolysate fractions (Fig. 1). The RP-HPLC-gradient applied: from 10 to 16% B over 2 min followed by 2 min isocratic elution; to 26% B over 8 min; to 35% B over 4 min; to 38% B in 32 min; then to 70% B over 3 min and "nishing with 5 min isocratic elution at 70% B. Peptide composition of the fractions categorised in groups I}V (Visser et al., 1989).
stated otherwise, a #ow rate of 60 ml\ min was applied. Detection was at 220 and 280 nm. The peptide composition of the bCN hydrolysate fractions (Fig. 1) as well as the IEC-puri"ed fractions were analysed by reversed-phase high-performance liquid-chromatography (RP-HPLC). The RP-HPLC
Measured value Peptide sequence (Da)
Calculated value (Da)
I
283.7 1012.5 747.4 779.3
106}107 106}113 108}113 170}176
283.3 1013.2 747.9 780.0
II
3478.0
1}28
3478.4
III
12216.0 12175.1 12480.0 12441.0 8755.7 8716.0 9021.3 8980.9
A (1}105) A (1}105) A (1}107) A (1}107) A (29}105) A (29}105) A (29}107) A (29}107)
12217.3 12177.3 12482.6 12442.6 8756.9 8716.9 9022.3 8982.2
IV
7355.8 8118.0 7091.0 7852.5 6360.7 7122.5 4483.4 3721.3
106}169 106}176 108}169 108}176 114}169 114}176 170}209 177}209
7357.6 8119.5 7092.2 7854.2 6362.3 7124.3 4484.4 3722.5
V
11823.0 11556.7 10827.1
106}209 108}209 114}209
11824.0 11558.6 10828.7
P.W.J.R. Caessens et al. / International Dairy Journal 9 (1999) 347}351
349
Table 2 Foam and emulsifying properties bCN hydrolysate fractions (Fig. 1). For conditions used see materials and methods section Sample
Process
Foam pH 4
Foam pH 6.7
Emulsion pH 4
Emulsion pH 6.7
b-casein
Formation Stabilisation
!!! !!!
## ###
n.d.
Floc
2.3 lm ###
SUP-1
Formation Stabilisation
## #
## #
n.d.
Floc
Coal
Coal
1.5 lm #
Formation Stabilisation
## #
Coal
## #
Coal
3 lm !
Cr/#oc
Formation Stabilisation
## !
## !!
n.d.
Floc
Coal
Coal
Formation Stabilisation
## #
Coal
## #
Coal
Formation Stabilisation
## !
Coal
## #
Coal
PEL-1
Formation Stabilisation
# ###
## ###
Floc
n.d.
SUP-4
Formation Stabilisation
### ###
## #
Floc Coal
1.7 lm #
PEL-4
Formation Stabilisation
### ###
## #
Floc Coal
n.d.
RET-1
PER-1
SUP-3
1.4 lm ## 1.5 lm !
Coal
n.d.
Floc
1.9 lm #
n.d.
Floc
1.7 lm !
Coal
n.d.
Floc
n.d.
Floc
n.d.
Floc
Floc
Floc
! and #indicate the extent of forming and stabilising property. n.d.: not determined. Coal: coalescence; #oc: #occulation; cr: creaming. Average particle size immediately after homogenising.
equipment used is described by Visser, Slangen and Rollema (1991). The gradient was formed with solvent A (0.1% tri#uoroacetic acid (TFA) in 10% v/v aqueous acetonitrile) and solvent B (0.07% v/v TFA in 90% aqueous acetonitrile) as indicated in the RP-HPLC "gures. Flow rate of 0.8 ml\ min was applied. Detection was at 220 nm using Turbochrom data-acquisition and processing software (Perkin-Elmer, Ueberlingen, Germany). Peptide components from interesting bCN-hydrolysate fractions were collected by preparative RP-HPLC and their masses were determined by electrospray-ionization mass-spectroscopy (ESI-MS) on a Quattro II triple quadrupole instrument (Micromass, Cheshire, UK). The raw mass spectral data were processed and transformed with the Masslynx software version 2.2 (Micromass). Peptide identi"cation was performed by combining the masses determined, the primary structure of the protein, and the known speci"city of plasmin. Foam, emulsion, and surface-active properties of the peptide fractions were determined in screening tests at pH 6.7 (pH of milk) and pH 4.0 (representative of acidic foods), k"0.075 and 203C (Caessens et al., 1997). Foam forming and stabilising ability of a protein solution
(0.01% w/v) was tested using a whipping method, and the foam height was monitored for 1 h. Emulsions (oil/water"1/9 v/v; 0.44% w/v protein) were made by homogenising in a laboratory high-pressure homogeniser, and the particle size distribution as well as the turbidity (at 0, 1 and 24 h) were measured. The in#uence of the various protein solutions (20 mg l\) on the surface pressure at the A/W interface was examined by measuring the rate of lowering the surface tension in a Langmuir trough. The screening tests used to investigate these functional properties are suitable to screen for di!erences in functionality between the various peptide fractions, although no information can be obtained concerning the mechanisms responsible for these functional properties.
3. Results and discussion Plasmin hydrolysis of bCN and fractionation of the hydrolysate, as outlined in Fig. 1, resulted in eight peptide fractions. The RP-HPLC patterns of these fractions is shown in Fig. 2. The peptides were categorised into "ve groups (I}V), indicated in Fig. 2, according to Visser, Noorman, Slangen and Rollema (1989). Table 1 shows
350
P.W.J.R. Caessens et al. / International Dairy Journal 9 (1999) 347}351
Fig. 3. IEC chromatogram (280 nm) of RET-1 (a) and RP-HPLC chromatograms of the IEC puri"ed fractions (b). The IEC-gradient applied: 5 min isocratic elution at 100% A; 2 min sample-injection (25 ml\ min) followed by elution as indicated. The RP-HPLC-gradient applied: from 25 to 30% B in 3 min followed by 6 min isocratic elution; to 36% B over 12 min; to 70% B over 5 min and "nishing with 5 min isocratic elution at 70% B.
the MS results determined for the several peak components present in groups I}V. With the mild hydrolysis conditions used bCN could be degraded into mainly three parts: hydrophobic fractions (PEL-1, SUP-4, and PEL-4), amphipathic fractions (SUP-1, SUP-2, SUP-3, and RET-1), and a strong hydrophilic fraction (PER-1). Table 2 emulsion properties of the eight fractions (Fig. 1). Most of the fractions dissolved well in the bu!ers used except for the hydrophobic fractions (PEL-1, SUP4, and PEL-4), which were poorly soluble at both pH values (as was bCN at pH 4.0). The inferior foam and emulsion-stabilising properties of PER-1 could be related to the low surface pressure induced by this fraction (results not shown). The small size of the peptide and its hydrophilicity could be additional reasons for its poor functional properties at both pH's tested (Turgeon, Gauthier, MolleH & LeH onil, 1992). At pH 6.7 and 203C the hydrophobic fractions (PEL-1, SUP-4, PEL-4) formed #occulated foams and emulsions,
possibly caused by strong hydrophobic interactions. However, when made at 43C (at which temperature the solubility of SUP-4 was increased, but PEL-1 and PEL-4 were still poorly soluble), emulsions of these fractions were also #occulated (results not shown). Also, undissolved particles of the hydrophobic fractions (present at pH 6.7) may cause bridging #occulation of the droplets or bubbles (Walstra, 1987). Alternatively, the hydrophobic peptides may provide an insu$cient repulsive or steric barrier between the emulsion droplets and/or foam bubbles to prevent #occulation. It appeared that the amphipathic fractions (SUP-1, SUP-2, SUP-3, and RET-1) lowered the surface tension, but not to the extent observed with bCN (and the hydrophobic fractions) at pH 6.7 (results not shown). The fractions were able to form and stabilise a foam, although coalescence occurred. The foam made with SUP-3 at pH 4.0 showed particularly rapid coalescence. At pH 6.7 the amphipathic fractions formed smaller emulsion droplets
P.W.J.R. Caessens et al. / International Dairy Journal 9 (1999) 347}351
than bCN. The emulsion stability of these fractions varied considerably. As SUP-3 (unstable emulsion) is a more puri"ed fraction than SUP-1, SUP-2, and RET-1 (stable emulsion), these results could suggest synergistic e!ects between peptides present in the latter fractions; similar results have been reported in the literature (Shimizu, Lee, Kaminogawa & Yamauchi, 1986; Lee, Shimizu, Kaminogawa & Yamauchi, 1987). However, this functionality could also be produced by one or more speci"c peptides of group III (RP-HPLC), present in RET-1 (and probably also in SUP-1 and SUP-2), but absent from SUP-3 (as determined with CE, results not shown). Therefore, RET-1 was further puri"ed by IEC. The peptide composition of the resulting IEC-fractions was analysed by RP-HPLC. Fig. 3 shows the IEC and RPHPLC results. MS analysis showed that IEC fraction-1 possessed mainly peptides from RP-HPLC group IV (Table 1); the main peptides present in IEC fraction-2 were bCN(f 29-105/107); whilst the main peptides present in IEC fraction-3 were bCN(f 1-105/107). Foam and emulsion properties of IEC fraction-2 [bCN(f 29-105/ 107)] and IEC fraction-3 [bCN(f 1-105/107)] were determined. The foam and emulsion forming properties of IEC fractions-2 and -3 were similar, but the emulsionstabilising properties di!ered: IEC fraction-2 had poor stabilising properties, whereas IEC fraction-3 had stabilising properties comparable with those of RET-1. It has been mentioned in the literature (Ter Beek, Ketelaars, McCain, Smulders, Walstra & Hemminga, 1996; Leaver & Dalgleish, 1990) that the hydrophilic N-terminus of bCN, when adsorbed onto an oil/water interface, is #exible and projects into the water phase. Apparently the hydrophilic N-terminus of the peptide is important for the stabilising e!ects, by causing electrosteric repulsion between the oil-droplets. This particular result demonstrates the role of a speci"c peptide sequence rather than some synergistic e!ects (see above) in the production of foaming and emulsifying properties. Further research will include detailed experiments to investigate the foam and emulsion properties of selected peptide fractions. Furthermore, secondary structure of peptides, both in solution and adsorbed at interfaces, will be analysed to elucidate structure-function relationships further. Acknowledgements Charles Slangen is thanked for performing the MS experiments. This research was funded by the IOP-Industrial Proteins and DMV-International.
351
References Andrews, A. T. (1978a). The composition, structure and origin of proteose-peptone component 5 of bovine milk. European Journal of Biochemistry, 90, 59}65. Andrews, A. T. (1978b). The composition, structure and origin of proteose-peptone component 8F of bovine milk. European Journal of Biochemistry, 90, 67}71. Arai, S., & Fujimaki, M. (1991). Enzymatic modi"cation of proteins with special reference to improving their functional properties. In P. F. Fox, Food enzymology*vol. 2, Chapter 20. London: Elsevier Science Publishers. Caessens, P. W. J. R., Gruppen, H., Visser, S., van Aken, G. A., & Voragen, A. G. J. (1997). Plasmin hydrolysis of b-casein: foam and emulsion properties of the fractionated hydrolysate. Journal of Agricultural and Food Chemistry, 45, 2935}2941. Eigel, W. N., Butler, J. E., ErnstroK m, C. A., Farrell Jr., H. M., Harwalkar, V. R., Jenness, R., Whitney, R., & McL (1984). Nomenclature of proteins of cow's milk: "fth revision. Journal of Dairy Science, 67, 1599}1631. Leaver, J., & Dalgleish, D. G. (1990). The topography of b-casein at an oil/water interface as determined from the kinetics of trypsin-catalysed hydrolysis. Biochimica et Biophysica Acta, 1041, 217}222. Lee, S. W., Shimizu, M., Kaminogawa, S., & Yamauchi, K. (1987). Emulsifying properties of a mixture of peptides derived from the enzymatic hydrolysates of bovine caseins. Agricultural and Biological Chemistry, 51, 1535}1540. Panyam, D., & Kilara, A. (1996). Enhancing the functionality of food proteins by enzymic modi"cation. Trends in Food Science and Technology, 7, 120}125. Phillips, L. G., Whitehead, D. M., & Kinsella, J. (1994). Structurefunction properties of food proteins. San Diego: Academic Press. Shimizu, M., Lee, S. W., Kaminogawa, S., & Yamauchi, K. (1986). Functional properties of a peptide of 23 residues of a -casein: changes in the emulsifying activity during puri"cation of the peptide. Journal of Food Science, 51, 1248}1252. Ter Beek, L. C., Ketelaars, M., McCain, D. C., Smulders, P. E. A., Walstra, P., & Hemminga, M. A. (1996). Nuclear magnetic resonance study of the conformation and dynamics of b-casein at the oil/water interface in emulsions. Biophysical Journal, 70, 2396}2402. Turgeon, S. L., Gauthier, S. F., MolleH , D., & LeH onil, J. (1992). Interfacial properties of tryptic peptides of b-lactoglobulin. Journal of Agricultural and Food Chemistry, 40, 669}675. Visser, S., Noorman, H. J., Slangen, K. J., & Rollema, H. S. (1989). Action of plasmin on bovine b-casein in a membrane reactor. Journal of Dairy Research, 56, 323}333. Visser, S., Slangen, K. J., & Rollema, H. S. (1991). Phenotyping of bovine milk proteins by reversed-phase high-performance liquid chromatography. Journal of Chromatography, 548, 361}370. Walstra, P. (1987). Overview of emulsion and foam stability. In E. Dickinson, Food emulsions and foams (pp. 242}257). London: Royal Society of Chemistry. Wilson, M., Mulvihill, D. M., Donnelly, W. J., & Gill, B. P. (1989). Surface-active properties at the air-water interface of b-casein and its fragments derived by plasmin proteolysis. Journal of Dairy Research, 56, 487}494.
Emulsion and foam properties of plasmin derived b-casein peptides P.W.J.R. Caessens , S. Visser, H. Gruppen , G.A. van Aken, A.G.J. Voragen * Department of Food Science, Wageningen Agricultural University, P.O. Box 8129, 6700 EV, Wageningen, The Netherlands Department of Biophysical Chemistry, Netherlands Institute for Dairy Research (NIZO), P.O. Box 20, 6710 BA, Ede, The Netherlands
Abstract In the present study the in#uence of plasmin hydrolysis of b-casein (bCN) on the foam and emulsion properties was tested. The hydrophilic, amphipathic and hydrophobic fractions produced by plasmin hydrolysis of bCN and fractionation of the hydrolysate, show clear di!erences in emulsion, foam and surface-active properties. The hydrophilic peptide possessed poor functional properties. The hydrophobic peptides from bCN showed interesting foam properties, especially at acidic pH. The amphipathic peptides exhibited improved emulsion-forming properties, compared to intact bCN. It seemed that the presence of the hydrophilic N-terminus (i.e. fragment 1}28) in these amphipathic peptides was important for their stabilising properties. 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Milk proteins are well known for their surface-active behaviour and their ability to form and stabilise foam and emulsions. Chemical, physical, enzymatic, and genetic modi"cation can be applied to alter the functionality of proteins (Phillips, Whitehead & Kinsella, 1994). Enzymatic modi"cations have the advantage of mild reaction conditions (Arai & Fujimaki, 1991; Phillips et al., 1994), and can be used to enhance some functionalities of food proteins (Panyam & Kilara, 1996). Plasmin is a proteolytic enzyme with a preference for Lys}X and Arg}X bonds. In fresh milk it converts b-casein (bCN) into the c-caseins, and to the complementary parts called proteose peptones (Andrews, 1978a,b; Eigel et al., 1984). Wilson, Mulvihill, Donnelly and Gill (1989) used plasmin to modify bCN to yield c-caseins and proteose peptones. The surface pressure at the air/water (A/W) interface induced by these hydrolysis products appeared to be di!erent from that of the intact protein. Therefore, it is likely that the foaming and emulsifying properties of bCN will be altered by plasmin hydrolysis as well. The objective of the present study was
* Corresponding author. Tel.: #31-317-48-32-09; fax#31-317-4848-93. E-mail address: [email protected] (A.G.J. Voragen)
to produce, by means of plasmin hydrolysis, peptides from bCN with altered foam and emulsion (and surfaceactive) properties, with the aim of identifying structurefunction relationships.
2. Material and methods Bovine bCN (90% w/w bCN based on dry weight) was purchased from Eurial (Rennes, France). Bovine plasmin (EC 3.4.21.7) was obtained from Sigma (St. Louis, USA; product number P-7911). Unless stated otherwise, all other chemicals were of analytical grade, and were purchased from Merck (Darmstadt, Germany) or BDH (Poole, UK). Plasmin hydrolysis of bCN and fractionation of the hydrolysate have been described elsewhere (Caessens, Gruppen, Visser, van Aken & Voragen, 1997). An outline of the hydrolysis and fractionation conditions applied is shown in Fig. 1. All peptide fractions were lyophilised and stored at 43C before further analysis. Ion-exchange chromatography (IEC) was performed on the AG KTA-explorer, controlled by a UNICORNcontrol system (Pharmacia Biotech, Uppsala, Sweden) using a SourceQ column (280 ml bed volume; Pharmacia). The gradient formed with solvent A (20 mM Tris/ HCl, pH 8) and solvent B (20 mM Tris/HCl, 1 M NaCl, pH 8) is given in the IEC-"gure shown. Except where
0958-6946/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 0 8 6 - 2
348
P.W.J.R. Caessens et al. / International Dairy Journal 9 (1999) 347}351
Fig. 1. Outline of the bCN hydrolysis and fractionation of the hydrolysate. Table 1 MS results for the RP-HPLC collected peaks of bCN/plasmin hydrolysates, categorized in RP-HPLC groups I}V (see Fig. 2) RP-HPLC groups
Fig. 2. RP-HPLC chromatograms of the bCN hydrolysate fractions (Fig. 1). The RP-HPLC-gradient applied: from 10 to 16% B over 2 min followed by 2 min isocratic elution; to 26% B over 8 min; to 35% B over 4 min; to 38% B in 32 min; then to 70% B over 3 min and "nishing with 5 min isocratic elution at 70% B. Peptide composition of the fractions categorised in groups I}V (Visser et al., 1989).
stated otherwise, a #ow rate of 60 ml\ min was applied. Detection was at 220 and 280 nm. The peptide composition of the bCN hydrolysate fractions (Fig. 1) as well as the IEC-puri"ed fractions were analysed by reversed-phase high-performance liquid-chromatography (RP-HPLC). The RP-HPLC
Measured value Peptide sequence (Da)
Calculated value (Da)
I
283.7 1012.5 747.4 779.3
106}107 106}113 108}113 170}176
283.3 1013.2 747.9 780.0
II
3478.0
1}28
3478.4
III
12216.0 12175.1 12480.0 12441.0 8755.7 8716.0 9021.3 8980.9
A (1}105) A (1}105) A (1}107) A (1}107) A (29}105) A (29}105) A (29}107) A (29}107)
12217.3 12177.3 12482.6 12442.6 8756.9 8716.9 9022.3 8982.2
IV
7355.8 8118.0 7091.0 7852.5 6360.7 7122.5 4483.4 3721.3
106}169 106}176 108}169 108}176 114}169 114}176 170}209 177}209
7357.6 8119.5 7092.2 7854.2 6362.3 7124.3 4484.4 3722.5
V
11823.0 11556.7 10827.1
106}209 108}209 114}209
11824.0 11558.6 10828.7
P.W.J.R. Caessens et al. / International Dairy Journal 9 (1999) 347}351
349
Table 2 Foam and emulsifying properties bCN hydrolysate fractions (Fig. 1). For conditions used see materials and methods section Sample
Process
Foam pH 4
Foam pH 6.7
Emulsion pH 4
Emulsion pH 6.7
b-casein
Formation Stabilisation
!!! !!!
## ###
n.d.
Floc
2.3 lm ###
SUP-1
Formation Stabilisation
## #
## #
n.d.
Floc
Coal
Coal
1.5 lm #
Formation Stabilisation
## #
Coal
## #
Coal
3 lm !
Cr/#oc
Formation Stabilisation
## !
## !!
n.d.
Floc
Coal
Coal
Formation Stabilisation
## #
Coal
## #
Coal
Formation Stabilisation
## !
Coal
## #
Coal
PEL-1
Formation Stabilisation
# ###
## ###
Floc
n.d.
SUP-4
Formation Stabilisation
### ###
## #
Floc Coal
1.7 lm #
PEL-4
Formation Stabilisation
### ###
## #
Floc Coal
n.d.
RET-1
PER-1
SUP-3
1.4 lm ## 1.5 lm !
Coal
n.d.
Floc
1.9 lm #
n.d.
Floc
1.7 lm !
Coal
n.d.
Floc
n.d.
Floc
n.d.
Floc
Floc
Floc
! and #indicate the extent of forming and stabilising property. n.d.: not determined. Coal: coalescence; #oc: #occulation; cr: creaming. Average particle size immediately after homogenising.
equipment used is described by Visser, Slangen and Rollema (1991). The gradient was formed with solvent A (0.1% tri#uoroacetic acid (TFA) in 10% v/v aqueous acetonitrile) and solvent B (0.07% v/v TFA in 90% aqueous acetonitrile) as indicated in the RP-HPLC "gures. Flow rate of 0.8 ml\ min was applied. Detection was at 220 nm using Turbochrom data-acquisition and processing software (Perkin-Elmer, Ueberlingen, Germany). Peptide components from interesting bCN-hydrolysate fractions were collected by preparative RP-HPLC and their masses were determined by electrospray-ionization mass-spectroscopy (ESI-MS) on a Quattro II triple quadrupole instrument (Micromass, Cheshire, UK). The raw mass spectral data were processed and transformed with the Masslynx software version 2.2 (Micromass). Peptide identi"cation was performed by combining the masses determined, the primary structure of the protein, and the known speci"city of plasmin. Foam, emulsion, and surface-active properties of the peptide fractions were determined in screening tests at pH 6.7 (pH of milk) and pH 4.0 (representative of acidic foods), k"0.075 and 203C (Caessens et al., 1997). Foam forming and stabilising ability of a protein solution
(0.01% w/v) was tested using a whipping method, and the foam height was monitored for 1 h. Emulsions (oil/water"1/9 v/v; 0.44% w/v protein) were made by homogenising in a laboratory high-pressure homogeniser, and the particle size distribution as well as the turbidity (at 0, 1 and 24 h) were measured. The in#uence of the various protein solutions (20 mg l\) on the surface pressure at the A/W interface was examined by measuring the rate of lowering the surface tension in a Langmuir trough. The screening tests used to investigate these functional properties are suitable to screen for di!erences in functionality between the various peptide fractions, although no information can be obtained concerning the mechanisms responsible for these functional properties.
3. Results and discussion Plasmin hydrolysis of bCN and fractionation of the hydrolysate, as outlined in Fig. 1, resulted in eight peptide fractions. The RP-HPLC patterns of these fractions is shown in Fig. 2. The peptides were categorised into "ve groups (I}V), indicated in Fig. 2, according to Visser, Noorman, Slangen and Rollema (1989). Table 1 shows
350
P.W.J.R. Caessens et al. / International Dairy Journal 9 (1999) 347}351
Fig. 3. IEC chromatogram (280 nm) of RET-1 (a) and RP-HPLC chromatograms of the IEC puri"ed fractions (b). The IEC-gradient applied: 5 min isocratic elution at 100% A; 2 min sample-injection (25 ml\ min) followed by elution as indicated. The RP-HPLC-gradient applied: from 25 to 30% B in 3 min followed by 6 min isocratic elution; to 36% B over 12 min; to 70% B over 5 min and "nishing with 5 min isocratic elution at 70% B.
the MS results determined for the several peak components present in groups I}V. With the mild hydrolysis conditions used bCN could be degraded into mainly three parts: hydrophobic fractions (PEL-1, SUP-4, and PEL-4), amphipathic fractions (SUP-1, SUP-2, SUP-3, and RET-1), and a strong hydrophilic fraction (PER-1). Table 2 emulsion properties of the eight fractions (Fig. 1). Most of the fractions dissolved well in the bu!ers used except for the hydrophobic fractions (PEL-1, SUP4, and PEL-4), which were poorly soluble at both pH values (as was bCN at pH 4.0). The inferior foam and emulsion-stabilising properties of PER-1 could be related to the low surface pressure induced by this fraction (results not shown). The small size of the peptide and its hydrophilicity could be additional reasons for its poor functional properties at both pH's tested (Turgeon, Gauthier, MolleH & LeH onil, 1992). At pH 6.7 and 203C the hydrophobic fractions (PEL-1, SUP-4, PEL-4) formed #occulated foams and emulsions,
possibly caused by strong hydrophobic interactions. However, when made at 43C (at which temperature the solubility of SUP-4 was increased, but PEL-1 and PEL-4 were still poorly soluble), emulsions of these fractions were also #occulated (results not shown). Also, undissolved particles of the hydrophobic fractions (present at pH 6.7) may cause bridging #occulation of the droplets or bubbles (Walstra, 1987). Alternatively, the hydrophobic peptides may provide an insu$cient repulsive or steric barrier between the emulsion droplets and/or foam bubbles to prevent #occulation. It appeared that the amphipathic fractions (SUP-1, SUP-2, SUP-3, and RET-1) lowered the surface tension, but not to the extent observed with bCN (and the hydrophobic fractions) at pH 6.7 (results not shown). The fractions were able to form and stabilise a foam, although coalescence occurred. The foam made with SUP-3 at pH 4.0 showed particularly rapid coalescence. At pH 6.7 the amphipathic fractions formed smaller emulsion droplets
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than bCN. The emulsion stability of these fractions varied considerably. As SUP-3 (unstable emulsion) is a more puri"ed fraction than SUP-1, SUP-2, and RET-1 (stable emulsion), these results could suggest synergistic e!ects between peptides present in the latter fractions; similar results have been reported in the literature (Shimizu, Lee, Kaminogawa & Yamauchi, 1986; Lee, Shimizu, Kaminogawa & Yamauchi, 1987). However, this functionality could also be produced by one or more speci"c peptides of group III (RP-HPLC), present in RET-1 (and probably also in SUP-1 and SUP-2), but absent from SUP-3 (as determined with CE, results not shown). Therefore, RET-1 was further puri"ed by IEC. The peptide composition of the resulting IEC-fractions was analysed by RP-HPLC. Fig. 3 shows the IEC and RPHPLC results. MS analysis showed that IEC fraction-1 possessed mainly peptides from RP-HPLC group IV (Table 1); the main peptides present in IEC fraction-2 were bCN(f 29-105/107); whilst the main peptides present in IEC fraction-3 were bCN(f 1-105/107). Foam and emulsion properties of IEC fraction-2 [bCN(f 29-105/ 107)] and IEC fraction-3 [bCN(f 1-105/107)] were determined. The foam and emulsion forming properties of IEC fractions-2 and -3 were similar, but the emulsionstabilising properties di!ered: IEC fraction-2 had poor stabilising properties, whereas IEC fraction-3 had stabilising properties comparable with those of RET-1. It has been mentioned in the literature (Ter Beek, Ketelaars, McCain, Smulders, Walstra & Hemminga, 1996; Leaver & Dalgleish, 1990) that the hydrophilic N-terminus of bCN, when adsorbed onto an oil/water interface, is #exible and projects into the water phase. Apparently the hydrophilic N-terminus of the peptide is important for the stabilising e!ects, by causing electrosteric repulsion between the oil-droplets. This particular result demonstrates the role of a speci"c peptide sequence rather than some synergistic e!ects (see above) in the production of foaming and emulsifying properties. Further research will include detailed experiments to investigate the foam and emulsion properties of selected peptide fractions. Furthermore, secondary structure of peptides, both in solution and adsorbed at interfaces, will be analysed to elucidate structure-function relationships further. Acknowledgements Charles Slangen is thanked for performing the MS experiments. This research was funded by the IOP-Industrial Proteins and DMV-International.
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