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Influence of glycosylation on foaming properties of bovine caseinomacropeptide
International Dairy Journal 19 (2009) 715–720
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International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Influence of glycosylation on foaming properties of bovine caseinomacropeptide Markus Kreuß a, *, Ingolf Krause b, Ulrich Kulozik a, c a
¨t Mu ¨ nchen, Chair for Food Process Engineering and Dairy Technology, Weihenstephaner Berg 1, 85354 Freising, Germany Technische Universita ¨ t Mu ¨ nchen, ZIEL Research Center for Nutrition and Food Science, Biochemistry Unit, Am Forum 5, 85354 Freising, Germany Technische Universita c ¨t Mu ¨ nchen, ZIEL Research Center for Nutrition and Food Science, Technology Unit, Weihenstephaner Berg 1, 85354 Freising, Germany Technische Universita b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 January 2009 Received in revised form 18 June 2009 Accepted 19 June 2009
Bovine caseinomacropeptide (CMP) fractions, both glycosylated (gCMP) and non-glycosylated (aCMP), were studied in detail for their foaming properties. The aCMP-stabilised foams showed significantly higher foam rigidity and stability than foams stabilised with gCMP, whereas both fractions yielded a high foaming ability with overruns of around 600%. The gCMP-stabilised foams, in particular, were considerably influenced by pH and showed reduced foaming properties above the pI, but superior properties at strong acidic pH below the pI. This influence was less significant for aCMP. An increase in ionic strength did not appear to influence either fraction. The combination of electrical, steric and hydrophilic barriers caused by the glycosylation of gCMP appears not to allow an ordered adsorption at the air/water interface, whereas aCMP can build a stable network at the surface. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Foaming is an important unit operation in the processing of foods. Proteins and peptides play a major role in forming and stabilising foams in aerated food products. Factors like heat treatment, pH and ionic environment have a significant influence on the foaming properties of milk proteins, as reported in detail by a number of authors (Campbell & Mougeot, 1999; Davis & Foegeding, 2007; Murray, 2007; Rodriguez Patino, Carrera Sanchez, & Rodriguez Nino, 2008; Rullier, Novales, & Axelos, 2008; Zhang & Goff, 2004; Zhang, Dalgleish, & Goff, 2004). Whereas small molecular mass surfactants rapidly decrease the surface tension, proteins adsorb slowly, but strongly at air/water interfaces (Dickinson, 1999). Regarding protein structure, it was shown that flexible proteins/peptides with high surface activity adsorb more rapidly to the interface than compact globular proteins (Martin, Grolle, Bos, Cohen-Stuart, & van Vliet, 2002). This study deals with bovine caseinomacropeptide (CMP), which is an example of a highly flexible peptide. CMP is the hydrophilic part of k-casein f(106–169), which is released by the endopeptidase chymosin during the renneting of milk. In contrast to globular proteins, CMP is an intrinsically disordered peptide (random coil) without a secondary or tertiary structure. This ‘‘unfolded state’’ of CMP is ensured by a low mean hydrophobicity, a high net charge and a high conformational flexibility (Uversky,
* Corresponding author. Tel.: þ49 8161 715032; fax: þ49 8161 714384. E-mail address: [email protected] (M. Kreuß). 0958-6946/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2009.06.012
2002). Concerning the surface and foaming properties of CMP, only one study has been published so far (Marshall, 1991). In this work a 10% CMP solution was found to have a higher overrun, but inferior foam stability compared with egg white. CMP foams in real systems like meringues were not stable and resulted in low meringue density when cooked. The previously reported studies were conducted with whole CMP. It is known that CMP is a highly diverse, heterogenic group of molecules due to its genetic variants as well as post-translational modifications. In other words, CMP is a class of peptides rather than one well defined individual molecule. The variation with two major genetic variants A and B from a total of 11 genetic variants (Buchberger & Dovc, 2000) is further amplified by a high and variable degree of glycosylation. Five different O-glycans that are covalently bound to threonine residues (T131, T133, T142 and T145) or serine (S165) have been identified so far (Holland, Deeth, & Alewood, 2006). In most cases, the highly negatively charged sialic acid (Nacetyl neuraminic acid) is the terminal carbohydrate. On the basis of glycosylation, CMP can be classified in two major fractions: the glycosylated gCMP and the non-glycosylated aCMP (aglyco-CMP). Although it can be expected that the biofunctional effects of gCMP, as reviewed by Thoma¨-Worringer, Sorensen, and LopezFandino (2006), might be stronger than that of aCMP, the correlation between the state of glycosylation and technofunctional properties, such as foaming, is not known. Only a few studies report on the effect of glycosylation on surface activity of proteins or peptides used as food product structuring agents. Such an effect on the formation and stabilisation of food foams has so far been observed in the foams of sparkling wines (Gonzalez-Ramos & Gonzalez, 2006; Nunez,
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Carrascosa, Gonzalez, Polo, & Martinez-Rodriguez, 2006). Improved emulsifying properties of artificially glycosylated ovalbumin with dextran and glucuronic acid were reported by Aoki et al. (1999) and Kato, Murata, and Kobayashi (1988). A single piece of evidence for milk proteins was given by Lemeste et al. (1990), who made observations that high levels of (artificial) glycosylation of caseins with galactose induced increased protein flexibility and improved foaming capacity. As a wide range of milk proteins, as well as other food proteins, are abundantly or partially glycosylated, it is of fundamental interest to obtain a deeper insight into the structure-function relationship of non-artificial glycoconjugates at hydrophobic/ hydrophilic interfaces, such as foams. Consequently, the present work is an approach to close this gap through a detailed study on the surface and foaming properties of CMP as a function of glycosylation. First, results of surface tension experiments of aCMP versus gCMP are given. Thereafter, detailed results of the foaming properties of aCMP and gCMP as a function of concentration, pH and ionic strength are presented. It should be mentioned that there are several additional possible techniques, such as surface rheology, thin film studies, foam bubble characterisation and X-ray tomography summarised in the reviews of Dickinson (1999) and Murray (2007). Those techniques can be applied in the future for further evaluation of the influence of a glycosylation on surface and foaming properties of unstructured peptides, such as CMP. 2. Experimental 2.1. Materials Native CMP fractions (aCMP and gCMP) were prepared by a membrane-adsorption-chromatography process according to Kreub, Krause, and Kulozik (2008). The purity of the gCMP and of the aGMP was 97.1% and 89.3%, respectively. Whey protein isolate (WPI BiPRO) was purchased from Davisco Foods International Inc. (Eden Prairie, MN, USA); it had a purity of 92.0% and comprised 20.8% a-lactalbumin and 71.2% b-lactoglobulin. Sodium acetate (NaAc), sodium chloride, hydrochloric acid and sodium hydroxide were purchased from Sigma (Taufkirchen, Germany). All chemicals and reagents were of analytical grade purity and all samples were prepared with bidistilled water. 2.2. Measurements of surface tension
concentrations were varied between 0.25% and 3% (w/v). Ionic strength experiments were performed by adding between 50 and 300 mM NaCl at a constant pH of 6.0 and 1% peptide concentration. Prior to foaming, the samples were cooled to 4 C. The solutions (250 mL) were foamed by whipping with a Thermomix TM 21 mixer (Vorwerk & Co., Wuppertal, Germany). The whipping was performed using a butterfly device at 1000 rpm for 90 s. 2.4. Foaming ability After the foams were prepared, the measurement of overrun was carried out within 2 min. The foam was filled in a weighing beaker with a plastic spoon. The formation of entrapped air bubbles was carefully avoided and excess foam was removed with a metal spatula. The overrun (OR) was expressed as a percentage and calculated using the following formula at constant volume:
ORð%Þ ¼
md mf
mf
100
(1)
where md is the mass (g) of the unwhipped protein dispersion, mf is the mass (g) of the final whipped foam. 2.5. Foam rigidity To measure foam rigidity, a beaker was filled and placed directly under a Stevens Leatherhead Food Research Association (LFRA) texture analyser (Stevens & Son, St. Albans, UK). The foam sample was penetrated by a metal ring geometry at a constant velocity of 2 mm s1 for a distance of 20 mm as described by Bals and Kulozik (2003). The force of the foam against penetration was expressed in mN and represents the maximum force. 2.6. Foam stability Foam stability was measured by monitoring the drainage according to the method of Bals and Kulozik (2003). The drainage is the quantity of fluid released from the lamella of the foam structure. For these measurements, plastic cups were filled with foam and weighed. After storage for 30 min at ambient temperature, the drainage fluid was carefully decanted. The drainage DR (%) was calculated from the mass ratio of drained water to initial foam:
DR ð%Þ ¼ Wdr ð30 minÞ=Wf 100
(2)
An automated drop volume tensiometer TVT2 (Lauda, Ko¨nigshofen, Germany) was used in dynamic mode (interfacial tension as a function of time) for the measurements of the decrease of surface tension (g) in aqueous aCMP or gCMP solutions vs. air. All solutions containing the peptides in bidistilled water were solvated for at least 1 h for full hydration. For the concentration experiments, peptide concentration was varied between 1.0% and 5.0% (w/w) at pH 6.0. For the pH experiments, pH was varied between 2.0 and 8.0 at a CMP concentration of 1.0%. For experiments on the impact of ionic strength, this was increased up to 50, 100, 200 or 500 mM NaCl at pH 6.0 and 1.0% concentration. The pH was adjusted using HCl and NaOH without using a buffer system. 2.3. Preparation of foams Peptide solutions were prepared by dispersing aCMP, gCMP or WPI in 0.02 M NaAc buffer and stirred for at least 2 h to ensure full hydration. The pH was adjusted (to 2.0, 4.0, 6.0 and 8.0) using HCl or NaOH, while the ionic strength was kept constant at 10 mM for all pH-values by adding an adequate quantity of NaCl. To assess the effect of concentration on foaming properties, aCMP or gCMP
Fig. 1. Time dependent surface tension g (mN m1) of individual CMP fractions as a function of concentration at pH 6.0: (–C–), 1% aCMP; (–:–), 5% aCMP; ($$$B$$$), 1% gGMP; ($$$6$$$), 5% gGMP.
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57.3 mN m1 at 1% and 54.7 mN m1 at 5% were observed for gCMP at pH 6.0. Thus, the surface activity was the same for 1% aCMP as for 5% gCMP. These equilibrium values can be related to stability of foams, thus indicating an increased foaming stability for aCMP compared with gCMP. Furthermore, both fractions showed a very fast initial decrease of g. Such an initial rapid decrease of surface tension can be correlated with improved foaming properties (Wilde & Clark, 1996). This major difference is a key for the understanding of all results described later in this paper. The more hydrophobic and smaller aCMP showed an increased surface activity and, what is shown and discussed later in this manuscript, increased foaming properties. The strong negative charge as well as steric and hydrophilic repulsion of the glycan residues induces a decrease of adsorption of gCMP at the surface. Accordingly, the formation of a stable surface layer or peptide network is suppressed.
Fig. 2. Surface tension g (mN m1) of aCMP (–C–) and gGMP ($$$B$$$) at equilibrium as a function of pH at 20 C and 1% peptide concentration. Vertical bars indicate the standard deviation.
where Wd is the weight of the drainage (g) after 30 min and Wf is the weight (g) of the foam after whipping. Consequently, an increased drainage value is accompanied by a decreased foaming stability. 2.7. Statistical analysis Foaming experiments were carried out three times and the surface tension was measured twice per experiment. The significance of differences between the measured values (P < 0.05) was determined using Statgraphics software (Statistical Graphics Corporation, Rockville, MD, USA).
3.1.2. Surface tension as a function of pH Both fractions showed a significant change in surface activity as a function of pH. As shown in Fig. 2, the lowest g-values were observed near the specific pI of each fraction (4.15 for aCMP and 3.15 for gCMP). Protein adsorption at the interface is most rapid at the pI since electrostatic repulsion is minimised for charged proteins (Dickinson, 1999). Subsequently, an increased adsorption of the hydrophobic domains of the peptide chain on the hydrophobic surface (gas bubble) without electrostatic repulsion is enabled. By increasing the pH, the charge of the molecules becomes more strongly negative which leads to a destabilisation of the surface layer. This effect was more pronounced for gCMP, showing increased g-values of 66.0 mN m1 as compared with 59.0 mN m1 for aCMP at pH 8.0 (P < 0.05). This significant difference at pHvalues above the pI can be related to a strong influence of the negatively charged sialic acid residues. Below the pI, the electrical influence of the glycans of the gCMP is neglected, which explains the similar g-values of around 57.0 mN m1 for both fractions.
3. Results and discussion 3.1. Surface tension
3.2. Foaming properties
3.1.1. Surface tension as a function of concentration Adsorption rates at the air/water interface of the aCMP or gCMP solutions were measured by quantitatively assessing the rate of surface tension (g) decline. With increasing CMP concentration, surface tension at equilibrium decreased, as the free surface was covered faster by the molecules (Fig. 1). Whereas g decreased from 55.7 mN m1 at 1%–52.1 mN m1 at 5% for aCMP, values of
3.2.1. Effect of aCMP or gCMP concentration Fig. 3A shows the overrun, rigidity and drainage of aCMPstabilised foams as a function of peptide concentration at pH 6.0. The overrun was generally high, reaching values of around 600%. It was only slightly affected by an increase in concentration. In contrast, both the rigidity and drainage were significantly affected by the concentration of both CMP fractions. Results for gCMP are
Fig. 3. Overrun (%; dCd), rigidity (mN; —-—) and drainage (%; $$$A$$$) as a function of (A) aCMP and (B) gCMP concentration at pH 6.0 and 10.0 mM ionic strength. Vertical bars indicate the standard deviation.
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Table 1 Mean values and standard deviation (n ¼ 3) of overrun (OR), foam rigidity (FR) and drainage (DR) of foams stabilised with non-glycosylated (aCMP) or glycosylated (gCMP) caseinomacropeptide, or whey protein isolate (WPI) at a concentration of 3.0% at pH 6.0. Protein
OR (%)
FR (mN)
DR (%)
aCMP gCMP WPI
617.3 10.7 561.9 5.6 348.7 10.2
180.0 3.6 50.0 7.1 98.7 7.3
41.3 1.0 94.2 2.4 49.3 1.2
shown in Fig. 3B. For concentrations of 0.5, 1.0 and 2.0% the overrun averaged around 530% with a slightly increased value of 560% (P < 0.05) at a gCMP concentration of 3%. The rigidity was influenced by the peptide concentration from what can be considered as a typical effect at increased concentrations while a clear effect on the drainage was not observed. Comparing the two CMP fractions, the overrun was in the same range, reaching high values typical obtained for small unstructured proteins or peptides. In contrast, the rigidity and stability (drainage) of aCMP-stabilised foams were higher compared with gCMP. The foaming properties of aCMP were superior to WPI stabilised foams at a concentration of 3.0% and pH 6.0 (Table 1). The WPI foams were prepared using exactly the same operating conditions. The results for aCMP, gCMP and WPI shown in Table 1 confirm the hypothesis that smaller flexible proteins show a higher overrun when compared to larger, more globular proteins. As the foam stability of aCMP is higher compared with WPI, aCMP can be considered to be an excellent foaming agent. For both CMP fractions, the foam stability and rigidity in particular are influenced by the peptide concentration, whereas the influence on foaming ability (overrun) was minimal. The first step of surface stabilisation is the adsorption of proteins at interfaces (Martin et al., 2002), which is followed by the stabilisation of the surface. As the foaming ability is independent of the peptide concentration, the first step does not seem to be limiting for CMP. This behaviour is observed for small molecular mass surfactants and rather uncommon for peptides or proteins. A further explanation might be that only very low concentrations of both fractions of CMP are sufficient for a high foaming ability.
Fig. 5. Impact of pH on rigidity (mN) of aGMP (dC d) and gGMP ($$$C$$$) at 10.0 mM ionic strength and a concentration of 1%. Vertical bars indicate the standard deviation.
3.2.2. Influence of pH Electrostatic interactions play a major role in interfacial and foaming properties, which are basically influenced by pH and ionic strength. Considerable variations in the hydrophilic-lipophilic balance of a protein occur as a function of pH – as well as conformational changes – especially for disordered proteins.
As shown in Fig. 4, a change in foaming ability (overrun) for both fractions was only observed for gCMP above pH 6.0. The overrun showed high values of around 600% over the whole pH range. For gCMP a decrease to 480% was observed only at pH 8.0, while this was not the case for aCMP (P < 0.05), which can be explained by the strong negative charge of the glycans. The generally weak response to a variation in pH can be explained by the fast adsorption of intrinsically disordered peptides at surfaces due to their high flexibility. The rapid reduction in surface tension described in Section 3.1 results in an enhanced adsorption of proteins/peptides to an interface and thereby especially results in improved foaming ability (Wilde & Clark, 1996), which is independent of pH or ionic strength. Usually, a correlation with increased foaming ability near the isoelectric point due to a shielding of the electrostatic repulsion is expected, as reported for a wide range of proteins (Dickinson, 1999; Foegeding, Luck, & Davis, 2006). This effect was not observed for both CMP fractions. In contrast to the foaming ability, the foam stability and rigidity, especially of gCMP, were considerably influenced by the pH (Figs. 5 and 6). As shown in Fig. 5, a maximum rigidity was observed at pH 4.0 (near the pI) for both fractions. Below the pI, the rigidity for gCMP-stabilised foams was even higher (P < 0.05) compared with aCMP. Increasing the pH above the pI resulted in a strong decrease in foam rigidity, which was more
Fig. 4. Impact of pH on overrun (%) of aGMP (dCd) and gGMP ($$$C$$$) at 10.0 mM ionic strength and a concentration of 1%. Vertical bars indicate the standard deviation.
Fig. 6. Impact of pH on drainage (%) of aGMP (dCd) and gGMP ($$$C$$$) at 10.0 mM ionic strength and a concentration of 1%. Vertical bars indicate the standard deviation.
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Fig. 7. Overrun (%; A) and drainage (%; B) of aGMP (dCd) and gGMP ($$$C$$$) stabilised foams as a function of ionic strength at a peptide concentration of 1% and pH 6.0.
evident for gCMP than for aCMP. These results were consistent with the data obtained for drainage (Fig. 6). Foams stabilised with aCMP were most stable at acidic pH with values of around 50% and slightly increased up to 70% (P < 0.05) at pH 8.0. In contrast, gCMP foams showed stable foams only at pH 2.0. At higher pH, the gCMP foams were very unstable and some were collapsed immediately after whipping. The observed effects may be correlated to a minimum of net charge of the peptide around the pI. This contributes to improved surface properties, resulting in better foaming stability. Due to the loss of electrical repulsion, an increased adsorption of both fractions is possible. These observations are in accordance with results for a variety of milk proteins, such as caseins (Lemeste et al., 1990). 3.2.3. Influence of ionic strength Due to a shielding of the electrical charge of proteins or peptides at pH levels above or below their pI, in general, surface adsorption should be increased as a function of ionic strength. The results shown in Fig. 7 reveal that neither the foaming ability (overrun) (A), nor the stability (B) of aCMP or gCMP-stabilised foams were affected by NaCl addition up to 300 mM. While values for overrun ranged around 600% for both fractions, drainage values were around 60% for aCMP and 85% for gCMP at pH 6.0. At this pH, both fractions are strongly negatively charged and should be influenced by ionic strength. These results were confirmed by measurements of the surface tension as a function of ionic strength. The surface tension of both CMP fractions was not influenced by an increase of NaCl up to 500 mM (data not shown). For a large number of other proteins, a positive correlation between NaCl addition and foaming properties was observed, for example, for whey proteins (Davis & Foegeding, 2004; Nicorescu et al., 2008), caseins (Zhang et al., 2004) and egg white proteins (Davis & Foegeding, 2007; Raikos, Campbell, & Euston, 2007). Due to the loss of electrical repulsion, the foaming ability in particular is increased, whereas stability is less commonly influenced. Furthermore, globular proteins, especially b-lactoglobulin, are partially denatured and unfolded by adding salts (Zhang et al., 2004). The result is a more reactive open conformation with an increased surface activity. As CMP is a random coiled peptide, this salting effect cannot be estimated. A possible explanation might be found in the initial ionic strength for all CMP solutions set to 10 mM. It could be that at this ionic strength, CMP is not sensitive to changes in ionic strength. However, this explanation is insufficient, as other protein-stabilised foams are significantly affected with increasing ionic strength even at much higher levels (Nicorescu et al., 2008; Raikos et al., 2007; Zhang et al., 2004). A decrease of peptide solubility at higher ionic strength (Agyare, Addo, & Xiong, 2009) possibly counters the increased shielding of the CMP charge. Furthermore, CMP-stabilised foams
seem not to be adequately explained by Derjaguin–Landau– Verwey–Overbeek (DLVO) theory (Derjaguin, 1989; Overbeek, 1977) typically used to describe colloidal phenomena. The foaming ability and stability appear to be more influenced by hydrophobic and hydrophilic interactions, steric stabilisation and the high molecular flexibility of the CMP molecule – with or without the glycosylation. 4. Conclusions A detailed analysis of the influence of a natural glycosylation on the adsorption of proteins at an air/water interface has not been made so far. This study for different glycosylated CMP fractions revealed a significant decrease of surface tension reduction, foam rigidity and stability for glycosylated CMP (gCMP) as compared with non-glycosylated CMP (aCMP) at pH-values typically applied for the production of foods. A hypothesis can be that a combination of steric and electrostatic repulsion of the glycans is responsible for this difference. The foaming stability of aCMP was higher than for WPI stabilised foams, thus making aCMP an excellent foaming agent. By contrast, foaming ability seems not to be influenced by the glycosylation of unstructured peptides and resulted in high values for both CMP fractions which were superior when compared with WPI stabilised foams. Furthermore, gCMP seems to be more influenced by a variation in pH compared to aCMP, which can be related to the negative charge of the terminal sialic acid residues at pH-values above the pI. This strong negative charge inhibits the formation of a stable peptide network at the interface, which, in contrast, is more pronounced for aCMP. However, as the foaming properties and surface tensions of both fractions were not influenced by an increase in ionic strength, it could be proposed that CMP-stabilised foams cannot be described using DLVO theory, but instead by hydrophobic, hydrophilic and steric interactions. Apart from these first insights into the foaming properties of CMP, additional techniques, especially surface rheology and thin film studies, could be used in the future to shed more light on the interfacial behaviour of this peptide and the influence of its glycosylation. Nevertheless, some initial conclusions of practical importance can be drawn from these results. Whey protein products, such as whey powder, whey protein concentrate or WPI normally contain varying amounts of CMP and ratios of aCMP to gCMP. With increased knowledge of the amount and ratio of glycosylation, product performance can be predicted or used for better product standardisation and characterisation. Acknowledgments The authors would like to thank Annette Bruemmer-Rolf for her highly valued contribution in the experimental phase of the work.
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The authors are grateful for the financial support of the ‘‘Bundesministerium fu¨r Wirtschaft und Arbeit (BMWA)’’ in the ‘‘Arbeitsgemeinschaft industrieller Forschungs-vereinigungen’’ ‘‘Otto von Guericke’’ e.V. (AiF) and the ‘‘Forschungskreis der Erna¨hrungsindustrie (FEI)’’ within the project AiF-FV 15187 N. References Agyare, K. K., Addo, K., & Xiong, Y. L. (2009). Emulsifying and foaming properties of transglutaminase-treated wheat gluten hydrolysate as influenced by pH, temperature and salt. Food Hydrocolloids, 23, 72–81. Aoki, T., Hiidome, Y., Kitahata, K., Sugimoto, Y., Ibrahim, H. R., & Kato, Y. (1999). Improvement of heat stability and emulsifying activity of ovalbumin by conjugation with glucuronic acid through the Maillard reaction. Food Research International, 32, 129–133. Bals, A., & Kulozik, U. (2003). Effect of pre-heating on the foaming properties of whey protein isolate using a membrane foaming apparatus. International Dairy Journal, 13, 903–908. Buchberger, J., & Dovc, P. (2000). Lactoprotein genetic variants in cattle. Food Technology and Biotechnology, 38, 91–98. Campbell, G. M., & Mougeot, E. (1999). Creation and characterisation of aerated food products. Trends in Food Science and Technology, 10, 283–296. Davis, J. P., & Foegeding, E. A. (2004). Foaming and interfacial properties of polymerized whey protein isolate. Journal of Food Science, 69, 404–410. Davis, J. P., & Foegeding, E. A. (2007). Comparisons of the foaming and interfacial properties of whey protein isolate and egg white proteins. Colloids and Surfaces B: Biointerfaces, 54, 200–210. Derjaguin, B. V. (1989). Theory of stability of colloids and thin films. New York, NY, USA: Consultants Bureau. Dickinson, E. (1999). Adsorbed protein layers at fluid interfaces: interactions, structure and surface rheology. Colloids and Surfaces B: Biointerfaces, 15, 161–176. Foegeding, E. A., Luck, P. J., & Davis, J. P. (2006). Factors determining the physical properties of protein foams. Food Hydrocolloids, 20, 284–292. Gonzalez-Ramos, D., & Gonzalez, R. (2006). Genetic determinants of the release of mannoproteins of enological interest by Saccharomyces cerevisiae. Journal of Agricultural and Food Chemistry, 54, 9411–9416. Holland, J. W., Deeth, H. C., & Alewood, P. F. (2006). Resolution and characterisation of multiple isoforms of bovine kappa-casein by 2-DE following a reversible cysteine-tagging enrichment strategy. Proteomics, 6, 3087–3095. Kato, A., Murata, K., & Kobayashi, K. (1988). Preparation and characterization of ovalbumin-dextran conjugate having excellent emulsifying properties. Journal of Agricultural and Food Chemistry, 36, 421–425.
Kreuß, M., Krause, I., & Kulozik, U. (2008). Separation of a glycosylated and nonglycosylated fraction of caseinomacropeptide using different anion-exchange stationary phases. Journal of Chromatography A, 1208, 126–132. Lemeste, M., Colas, B., Simatos, D., Closs, B., Courthaudon, J. L., & Lorient, D. (1990). Contribution of protein flexibility to the foaming properties of casein. Journal of Food Science, 55, 1445–1447. Marshall, S. C. (1991). Casein macropeptide from whey – a new product opportunity. Food Research Quarterly, 51, 86–91. Martin, A. H., Grolle, K., Bos, M. A., Cohen-Stuart, M. A., & van Vliet, T. (2002). Network forming properties of various proteins adsorbed at the air/water interface in relation to foam stability. Journal of Colloid and Interface Science, 254, 173–178. Murray, B. S. (2007). Stabilization of bubbles and foams. Current Opinion in Colloid and Interface Science, 12, 232–241. Nicorescu, I., Loisel, C., Vial, C., Riaublanc, A., Djelveh, G., Cuvelier, G., et al. (2008). Combined effect of dynamic heat treatment and ionic strength on the properties of whey protein foams – part II. Food Research International, 41, 980–988. Nunez, Y. P., Carrascosa, A. V., Gonzalez, R., Polo, M. C., & Martinez-Rodriguez, A. (2006). Isolation and characterization of a thermally extracted yeast cell wall fraction potentially useful for improving the foaming properties of sparkling wines. Journal of Agricultural and Food Chemistry, 54, 7898–7903. Overbeek, J. Th. G. (1977). Recent developments in the understanding of colloid stability. In M. Kerker, A. C. Zettlemoyer, & R. L. Rowell (Eds.), Colloid and interface science (pp. 431–445). New York, NY, USA: Academic Press. Raikos, V., Campbell, L., & Euston, S. R. (2007). Effects of sucrose and sodium chloride on foaming properties of egg white proteins. Food Research International, 40, 347–355. Rodriguez Patino, J. M., Carrera Sanchez, C., & Rodriguez Nino, M. R. (2008). Implications of interfacial characteristics of food foaming agents in foam formulations. Advances in Colloid and Interface Science, 140, 95–113. Rullier, B., Novales, B., & Axelos, M. A. V. (2008). Effect of protein aggregates on foaming properties of b-lactoglobulin. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 330, 96–102. Thoma¨-Worringer, C., Sorensen, J., & Lopez-Fandino, R. (2006). Health effects and technological features of caseinomacropeptide. International Dairy Journal, 16, 1324–1333. Uversky, V. N. (2002). Natively unfolded proteins: a point where biology waits for physics. Protein Science, 11, 739–756. Wilde, P. J., & Clark, D. C. (1996). Foam formation and stability. InMethods of testing protein functionality, Vol 1 (pp. 110–152). London, UK: Blackie Academic. Zhang, Z., Dalgleish, D. G., & Goff, H. D. (2004). Effect of pH and ionic strength on competitive protein adsorption to air/water interfaces in aqueous foams made with mixed milk proteins. Colloids and Surfaces B: Biointerfaces, 34, 113–121. Zhang, Z., & Goff, H. D. (2004). Protein distribution at air interfaces in dairy foams and ice cream as affected by casein dissociation and emulsifiers. International Dairy Journal, 14, 647–657.
Contents lists available at ScienceDirect
International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Influence of glycosylation on foaming properties of bovine caseinomacropeptide Markus Kreuß a, *, Ingolf Krause b, Ulrich Kulozik a, c a
¨t Mu ¨ nchen, Chair for Food Process Engineering and Dairy Technology, Weihenstephaner Berg 1, 85354 Freising, Germany Technische Universita ¨ t Mu ¨ nchen, ZIEL Research Center for Nutrition and Food Science, Biochemistry Unit, Am Forum 5, 85354 Freising, Germany Technische Universita c ¨t Mu ¨ nchen, ZIEL Research Center for Nutrition and Food Science, Technology Unit, Weihenstephaner Berg 1, 85354 Freising, Germany Technische Universita b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 January 2009 Received in revised form 18 June 2009 Accepted 19 June 2009
Bovine caseinomacropeptide (CMP) fractions, both glycosylated (gCMP) and non-glycosylated (aCMP), were studied in detail for their foaming properties. The aCMP-stabilised foams showed significantly higher foam rigidity and stability than foams stabilised with gCMP, whereas both fractions yielded a high foaming ability with overruns of around 600%. The gCMP-stabilised foams, in particular, were considerably influenced by pH and showed reduced foaming properties above the pI, but superior properties at strong acidic pH below the pI. This influence was less significant for aCMP. An increase in ionic strength did not appear to influence either fraction. The combination of electrical, steric and hydrophilic barriers caused by the glycosylation of gCMP appears not to allow an ordered adsorption at the air/water interface, whereas aCMP can build a stable network at the surface. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Foaming is an important unit operation in the processing of foods. Proteins and peptides play a major role in forming and stabilising foams in aerated food products. Factors like heat treatment, pH and ionic environment have a significant influence on the foaming properties of milk proteins, as reported in detail by a number of authors (Campbell & Mougeot, 1999; Davis & Foegeding, 2007; Murray, 2007; Rodriguez Patino, Carrera Sanchez, & Rodriguez Nino, 2008; Rullier, Novales, & Axelos, 2008; Zhang & Goff, 2004; Zhang, Dalgleish, & Goff, 2004). Whereas small molecular mass surfactants rapidly decrease the surface tension, proteins adsorb slowly, but strongly at air/water interfaces (Dickinson, 1999). Regarding protein structure, it was shown that flexible proteins/peptides with high surface activity adsorb more rapidly to the interface than compact globular proteins (Martin, Grolle, Bos, Cohen-Stuart, & van Vliet, 2002). This study deals with bovine caseinomacropeptide (CMP), which is an example of a highly flexible peptide. CMP is the hydrophilic part of k-casein f(106–169), which is released by the endopeptidase chymosin during the renneting of milk. In contrast to globular proteins, CMP is an intrinsically disordered peptide (random coil) without a secondary or tertiary structure. This ‘‘unfolded state’’ of CMP is ensured by a low mean hydrophobicity, a high net charge and a high conformational flexibility (Uversky,
* Corresponding author. Tel.: þ49 8161 715032; fax: þ49 8161 714384. E-mail address: [email protected] (M. Kreuß). 0958-6946/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2009.06.012
2002). Concerning the surface and foaming properties of CMP, only one study has been published so far (Marshall, 1991). In this work a 10% CMP solution was found to have a higher overrun, but inferior foam stability compared with egg white. CMP foams in real systems like meringues were not stable and resulted in low meringue density when cooked. The previously reported studies were conducted with whole CMP. It is known that CMP is a highly diverse, heterogenic group of molecules due to its genetic variants as well as post-translational modifications. In other words, CMP is a class of peptides rather than one well defined individual molecule. The variation with two major genetic variants A and B from a total of 11 genetic variants (Buchberger & Dovc, 2000) is further amplified by a high and variable degree of glycosylation. Five different O-glycans that are covalently bound to threonine residues (T131, T133, T142 and T145) or serine (S165) have been identified so far (Holland, Deeth, & Alewood, 2006). In most cases, the highly negatively charged sialic acid (Nacetyl neuraminic acid) is the terminal carbohydrate. On the basis of glycosylation, CMP can be classified in two major fractions: the glycosylated gCMP and the non-glycosylated aCMP (aglyco-CMP). Although it can be expected that the biofunctional effects of gCMP, as reviewed by Thoma¨-Worringer, Sorensen, and LopezFandino (2006), might be stronger than that of aCMP, the correlation between the state of glycosylation and technofunctional properties, such as foaming, is not known. Only a few studies report on the effect of glycosylation on surface activity of proteins or peptides used as food product structuring agents. Such an effect on the formation and stabilisation of food foams has so far been observed in the foams of sparkling wines (Gonzalez-Ramos & Gonzalez, 2006; Nunez,
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Carrascosa, Gonzalez, Polo, & Martinez-Rodriguez, 2006). Improved emulsifying properties of artificially glycosylated ovalbumin with dextran and glucuronic acid were reported by Aoki et al. (1999) and Kato, Murata, and Kobayashi (1988). A single piece of evidence for milk proteins was given by Lemeste et al. (1990), who made observations that high levels of (artificial) glycosylation of caseins with galactose induced increased protein flexibility and improved foaming capacity. As a wide range of milk proteins, as well as other food proteins, are abundantly or partially glycosylated, it is of fundamental interest to obtain a deeper insight into the structure-function relationship of non-artificial glycoconjugates at hydrophobic/ hydrophilic interfaces, such as foams. Consequently, the present work is an approach to close this gap through a detailed study on the surface and foaming properties of CMP as a function of glycosylation. First, results of surface tension experiments of aCMP versus gCMP are given. Thereafter, detailed results of the foaming properties of aCMP and gCMP as a function of concentration, pH and ionic strength are presented. It should be mentioned that there are several additional possible techniques, such as surface rheology, thin film studies, foam bubble characterisation and X-ray tomography summarised in the reviews of Dickinson (1999) and Murray (2007). Those techniques can be applied in the future for further evaluation of the influence of a glycosylation on surface and foaming properties of unstructured peptides, such as CMP. 2. Experimental 2.1. Materials Native CMP fractions (aCMP and gCMP) were prepared by a membrane-adsorption-chromatography process according to Kreub, Krause, and Kulozik (2008). The purity of the gCMP and of the aGMP was 97.1% and 89.3%, respectively. Whey protein isolate (WPI BiPRO) was purchased from Davisco Foods International Inc. (Eden Prairie, MN, USA); it had a purity of 92.0% and comprised 20.8% a-lactalbumin and 71.2% b-lactoglobulin. Sodium acetate (NaAc), sodium chloride, hydrochloric acid and sodium hydroxide were purchased from Sigma (Taufkirchen, Germany). All chemicals and reagents were of analytical grade purity and all samples were prepared with bidistilled water. 2.2. Measurements of surface tension
concentrations were varied between 0.25% and 3% (w/v). Ionic strength experiments were performed by adding between 50 and 300 mM NaCl at a constant pH of 6.0 and 1% peptide concentration. Prior to foaming, the samples were cooled to 4 C. The solutions (250 mL) were foamed by whipping with a Thermomix TM 21 mixer (Vorwerk & Co., Wuppertal, Germany). The whipping was performed using a butterfly device at 1000 rpm for 90 s. 2.4. Foaming ability After the foams were prepared, the measurement of overrun was carried out within 2 min. The foam was filled in a weighing beaker with a plastic spoon. The formation of entrapped air bubbles was carefully avoided and excess foam was removed with a metal spatula. The overrun (OR) was expressed as a percentage and calculated using the following formula at constant volume:
ORð%Þ ¼
md mf
mf
100
(1)
where md is the mass (g) of the unwhipped protein dispersion, mf is the mass (g) of the final whipped foam. 2.5. Foam rigidity To measure foam rigidity, a beaker was filled and placed directly under a Stevens Leatherhead Food Research Association (LFRA) texture analyser (Stevens & Son, St. Albans, UK). The foam sample was penetrated by a metal ring geometry at a constant velocity of 2 mm s1 for a distance of 20 mm as described by Bals and Kulozik (2003). The force of the foam against penetration was expressed in mN and represents the maximum force. 2.6. Foam stability Foam stability was measured by monitoring the drainage according to the method of Bals and Kulozik (2003). The drainage is the quantity of fluid released from the lamella of the foam structure. For these measurements, plastic cups were filled with foam and weighed. After storage for 30 min at ambient temperature, the drainage fluid was carefully decanted. The drainage DR (%) was calculated from the mass ratio of drained water to initial foam:
DR ð%Þ ¼ Wdr ð30 minÞ=Wf 100
(2)
An automated drop volume tensiometer TVT2 (Lauda, Ko¨nigshofen, Germany) was used in dynamic mode (interfacial tension as a function of time) for the measurements of the decrease of surface tension (g) in aqueous aCMP or gCMP solutions vs. air. All solutions containing the peptides in bidistilled water were solvated for at least 1 h for full hydration. For the concentration experiments, peptide concentration was varied between 1.0% and 5.0% (w/w) at pH 6.0. For the pH experiments, pH was varied between 2.0 and 8.0 at a CMP concentration of 1.0%. For experiments on the impact of ionic strength, this was increased up to 50, 100, 200 or 500 mM NaCl at pH 6.0 and 1.0% concentration. The pH was adjusted using HCl and NaOH without using a buffer system. 2.3. Preparation of foams Peptide solutions were prepared by dispersing aCMP, gCMP or WPI in 0.02 M NaAc buffer and stirred for at least 2 h to ensure full hydration. The pH was adjusted (to 2.0, 4.0, 6.0 and 8.0) using HCl or NaOH, while the ionic strength was kept constant at 10 mM for all pH-values by adding an adequate quantity of NaCl. To assess the effect of concentration on foaming properties, aCMP or gCMP
Fig. 1. Time dependent surface tension g (mN m1) of individual CMP fractions as a function of concentration at pH 6.0: (–C–), 1% aCMP; (–:–), 5% aCMP; ($$$B$$$), 1% gGMP; ($$$6$$$), 5% gGMP.
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57.3 mN m1 at 1% and 54.7 mN m1 at 5% were observed for gCMP at pH 6.0. Thus, the surface activity was the same for 1% aCMP as for 5% gCMP. These equilibrium values can be related to stability of foams, thus indicating an increased foaming stability for aCMP compared with gCMP. Furthermore, both fractions showed a very fast initial decrease of g. Such an initial rapid decrease of surface tension can be correlated with improved foaming properties (Wilde & Clark, 1996). This major difference is a key for the understanding of all results described later in this paper. The more hydrophobic and smaller aCMP showed an increased surface activity and, what is shown and discussed later in this manuscript, increased foaming properties. The strong negative charge as well as steric and hydrophilic repulsion of the glycan residues induces a decrease of adsorption of gCMP at the surface. Accordingly, the formation of a stable surface layer or peptide network is suppressed.
Fig. 2. Surface tension g (mN m1) of aCMP (–C–) and gGMP ($$$B$$$) at equilibrium as a function of pH at 20 C and 1% peptide concentration. Vertical bars indicate the standard deviation.
where Wd is the weight of the drainage (g) after 30 min and Wf is the weight (g) of the foam after whipping. Consequently, an increased drainage value is accompanied by a decreased foaming stability. 2.7. Statistical analysis Foaming experiments were carried out three times and the surface tension was measured twice per experiment. The significance of differences between the measured values (P < 0.05) was determined using Statgraphics software (Statistical Graphics Corporation, Rockville, MD, USA).
3.1.2. Surface tension as a function of pH Both fractions showed a significant change in surface activity as a function of pH. As shown in Fig. 2, the lowest g-values were observed near the specific pI of each fraction (4.15 for aCMP and 3.15 for gCMP). Protein adsorption at the interface is most rapid at the pI since electrostatic repulsion is minimised for charged proteins (Dickinson, 1999). Subsequently, an increased adsorption of the hydrophobic domains of the peptide chain on the hydrophobic surface (gas bubble) without electrostatic repulsion is enabled. By increasing the pH, the charge of the molecules becomes more strongly negative which leads to a destabilisation of the surface layer. This effect was more pronounced for gCMP, showing increased g-values of 66.0 mN m1 as compared with 59.0 mN m1 for aCMP at pH 8.0 (P < 0.05). This significant difference at pHvalues above the pI can be related to a strong influence of the negatively charged sialic acid residues. Below the pI, the electrical influence of the glycans of the gCMP is neglected, which explains the similar g-values of around 57.0 mN m1 for both fractions.
3. Results and discussion 3.1. Surface tension
3.2. Foaming properties
3.1.1. Surface tension as a function of concentration Adsorption rates at the air/water interface of the aCMP or gCMP solutions were measured by quantitatively assessing the rate of surface tension (g) decline. With increasing CMP concentration, surface tension at equilibrium decreased, as the free surface was covered faster by the molecules (Fig. 1). Whereas g decreased from 55.7 mN m1 at 1%–52.1 mN m1 at 5% for aCMP, values of
3.2.1. Effect of aCMP or gCMP concentration Fig. 3A shows the overrun, rigidity and drainage of aCMPstabilised foams as a function of peptide concentration at pH 6.0. The overrun was generally high, reaching values of around 600%. It was only slightly affected by an increase in concentration. In contrast, both the rigidity and drainage were significantly affected by the concentration of both CMP fractions. Results for gCMP are
Fig. 3. Overrun (%; dCd), rigidity (mN; —-—) and drainage (%; $$$A$$$) as a function of (A) aCMP and (B) gCMP concentration at pH 6.0 and 10.0 mM ionic strength. Vertical bars indicate the standard deviation.
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Table 1 Mean values and standard deviation (n ¼ 3) of overrun (OR), foam rigidity (FR) and drainage (DR) of foams stabilised with non-glycosylated (aCMP) or glycosylated (gCMP) caseinomacropeptide, or whey protein isolate (WPI) at a concentration of 3.0% at pH 6.0. Protein
OR (%)
FR (mN)
DR (%)
aCMP gCMP WPI
617.3 10.7 561.9 5.6 348.7 10.2
180.0 3.6 50.0 7.1 98.7 7.3
41.3 1.0 94.2 2.4 49.3 1.2
shown in Fig. 3B. For concentrations of 0.5, 1.0 and 2.0% the overrun averaged around 530% with a slightly increased value of 560% (P < 0.05) at a gCMP concentration of 3%. The rigidity was influenced by the peptide concentration from what can be considered as a typical effect at increased concentrations while a clear effect on the drainage was not observed. Comparing the two CMP fractions, the overrun was in the same range, reaching high values typical obtained for small unstructured proteins or peptides. In contrast, the rigidity and stability (drainage) of aCMP-stabilised foams were higher compared with gCMP. The foaming properties of aCMP were superior to WPI stabilised foams at a concentration of 3.0% and pH 6.0 (Table 1). The WPI foams were prepared using exactly the same operating conditions. The results for aCMP, gCMP and WPI shown in Table 1 confirm the hypothesis that smaller flexible proteins show a higher overrun when compared to larger, more globular proteins. As the foam stability of aCMP is higher compared with WPI, aCMP can be considered to be an excellent foaming agent. For both CMP fractions, the foam stability and rigidity in particular are influenced by the peptide concentration, whereas the influence on foaming ability (overrun) was minimal. The first step of surface stabilisation is the adsorption of proteins at interfaces (Martin et al., 2002), which is followed by the stabilisation of the surface. As the foaming ability is independent of the peptide concentration, the first step does not seem to be limiting for CMP. This behaviour is observed for small molecular mass surfactants and rather uncommon for peptides or proteins. A further explanation might be that only very low concentrations of both fractions of CMP are sufficient for a high foaming ability.
Fig. 5. Impact of pH on rigidity (mN) of aGMP (dC d) and gGMP ($$$C$$$) at 10.0 mM ionic strength and a concentration of 1%. Vertical bars indicate the standard deviation.
3.2.2. Influence of pH Electrostatic interactions play a major role in interfacial and foaming properties, which are basically influenced by pH and ionic strength. Considerable variations in the hydrophilic-lipophilic balance of a protein occur as a function of pH – as well as conformational changes – especially for disordered proteins.
As shown in Fig. 4, a change in foaming ability (overrun) for both fractions was only observed for gCMP above pH 6.0. The overrun showed high values of around 600% over the whole pH range. For gCMP a decrease to 480% was observed only at pH 8.0, while this was not the case for aCMP (P < 0.05), which can be explained by the strong negative charge of the glycans. The generally weak response to a variation in pH can be explained by the fast adsorption of intrinsically disordered peptides at surfaces due to their high flexibility. The rapid reduction in surface tension described in Section 3.1 results in an enhanced adsorption of proteins/peptides to an interface and thereby especially results in improved foaming ability (Wilde & Clark, 1996), which is independent of pH or ionic strength. Usually, a correlation with increased foaming ability near the isoelectric point due to a shielding of the electrostatic repulsion is expected, as reported for a wide range of proteins (Dickinson, 1999; Foegeding, Luck, & Davis, 2006). This effect was not observed for both CMP fractions. In contrast to the foaming ability, the foam stability and rigidity, especially of gCMP, were considerably influenced by the pH (Figs. 5 and 6). As shown in Fig. 5, a maximum rigidity was observed at pH 4.0 (near the pI) for both fractions. Below the pI, the rigidity for gCMP-stabilised foams was even higher (P < 0.05) compared with aCMP. Increasing the pH above the pI resulted in a strong decrease in foam rigidity, which was more
Fig. 4. Impact of pH on overrun (%) of aGMP (dCd) and gGMP ($$$C$$$) at 10.0 mM ionic strength and a concentration of 1%. Vertical bars indicate the standard deviation.
Fig. 6. Impact of pH on drainage (%) of aGMP (dCd) and gGMP ($$$C$$$) at 10.0 mM ionic strength and a concentration of 1%. Vertical bars indicate the standard deviation.
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Fig. 7. Overrun (%; A) and drainage (%; B) of aGMP (dCd) and gGMP ($$$C$$$) stabilised foams as a function of ionic strength at a peptide concentration of 1% and pH 6.0.
evident for gCMP than for aCMP. These results were consistent with the data obtained for drainage (Fig. 6). Foams stabilised with aCMP were most stable at acidic pH with values of around 50% and slightly increased up to 70% (P < 0.05) at pH 8.0. In contrast, gCMP foams showed stable foams only at pH 2.0. At higher pH, the gCMP foams were very unstable and some were collapsed immediately after whipping. The observed effects may be correlated to a minimum of net charge of the peptide around the pI. This contributes to improved surface properties, resulting in better foaming stability. Due to the loss of electrical repulsion, an increased adsorption of both fractions is possible. These observations are in accordance with results for a variety of milk proteins, such as caseins (Lemeste et al., 1990). 3.2.3. Influence of ionic strength Due to a shielding of the electrical charge of proteins or peptides at pH levels above or below their pI, in general, surface adsorption should be increased as a function of ionic strength. The results shown in Fig. 7 reveal that neither the foaming ability (overrun) (A), nor the stability (B) of aCMP or gCMP-stabilised foams were affected by NaCl addition up to 300 mM. While values for overrun ranged around 600% for both fractions, drainage values were around 60% for aCMP and 85% for gCMP at pH 6.0. At this pH, both fractions are strongly negatively charged and should be influenced by ionic strength. These results were confirmed by measurements of the surface tension as a function of ionic strength. The surface tension of both CMP fractions was not influenced by an increase of NaCl up to 500 mM (data not shown). For a large number of other proteins, a positive correlation between NaCl addition and foaming properties was observed, for example, for whey proteins (Davis & Foegeding, 2004; Nicorescu et al., 2008), caseins (Zhang et al., 2004) and egg white proteins (Davis & Foegeding, 2007; Raikos, Campbell, & Euston, 2007). Due to the loss of electrical repulsion, the foaming ability in particular is increased, whereas stability is less commonly influenced. Furthermore, globular proteins, especially b-lactoglobulin, are partially denatured and unfolded by adding salts (Zhang et al., 2004). The result is a more reactive open conformation with an increased surface activity. As CMP is a random coiled peptide, this salting effect cannot be estimated. A possible explanation might be found in the initial ionic strength for all CMP solutions set to 10 mM. It could be that at this ionic strength, CMP is not sensitive to changes in ionic strength. However, this explanation is insufficient, as other protein-stabilised foams are significantly affected with increasing ionic strength even at much higher levels (Nicorescu et al., 2008; Raikos et al., 2007; Zhang et al., 2004). A decrease of peptide solubility at higher ionic strength (Agyare, Addo, & Xiong, 2009) possibly counters the increased shielding of the CMP charge. Furthermore, CMP-stabilised foams
seem not to be adequately explained by Derjaguin–Landau– Verwey–Overbeek (DLVO) theory (Derjaguin, 1989; Overbeek, 1977) typically used to describe colloidal phenomena. The foaming ability and stability appear to be more influenced by hydrophobic and hydrophilic interactions, steric stabilisation and the high molecular flexibility of the CMP molecule – with or without the glycosylation. 4. Conclusions A detailed analysis of the influence of a natural glycosylation on the adsorption of proteins at an air/water interface has not been made so far. This study for different glycosylated CMP fractions revealed a significant decrease of surface tension reduction, foam rigidity and stability for glycosylated CMP (gCMP) as compared with non-glycosylated CMP (aCMP) at pH-values typically applied for the production of foods. A hypothesis can be that a combination of steric and electrostatic repulsion of the glycans is responsible for this difference. The foaming stability of aCMP was higher than for WPI stabilised foams, thus making aCMP an excellent foaming agent. By contrast, foaming ability seems not to be influenced by the glycosylation of unstructured peptides and resulted in high values for both CMP fractions which were superior when compared with WPI stabilised foams. Furthermore, gCMP seems to be more influenced by a variation in pH compared to aCMP, which can be related to the negative charge of the terminal sialic acid residues at pH-values above the pI. This strong negative charge inhibits the formation of a stable peptide network at the interface, which, in contrast, is more pronounced for aCMP. However, as the foaming properties and surface tensions of both fractions were not influenced by an increase in ionic strength, it could be proposed that CMP-stabilised foams cannot be described using DLVO theory, but instead by hydrophobic, hydrophilic and steric interactions. Apart from these first insights into the foaming properties of CMP, additional techniques, especially surface rheology and thin film studies, could be used in the future to shed more light on the interfacial behaviour of this peptide and the influence of its glycosylation. Nevertheless, some initial conclusions of practical importance can be drawn from these results. Whey protein products, such as whey powder, whey protein concentrate or WPI normally contain varying amounts of CMP and ratios of aCMP to gCMP. With increased knowledge of the amount and ratio of glycosylation, product performance can be predicted or used for better product standardisation and characterisation. Acknowledgments The authors would like to thank Annette Bruemmer-Rolf for her highly valued contribution in the experimental phase of the work.
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The authors are grateful for the financial support of the ‘‘Bundesministerium fu¨r Wirtschaft und Arbeit (BMWA)’’ in the ‘‘Arbeitsgemeinschaft industrieller Forschungs-vereinigungen’’ ‘‘Otto von Guericke’’ e.V. (AiF) and the ‘‘Forschungskreis der Erna¨hrungsindustrie (FEI)’’ within the project AiF-FV 15187 N. References Agyare, K. K., Addo, K., & Xiong, Y. L. (2009). Emulsifying and foaming properties of transglutaminase-treated wheat gluten hydrolysate as influenced by pH, temperature and salt. Food Hydrocolloids, 23, 72–81. Aoki, T., Hiidome, Y., Kitahata, K., Sugimoto, Y., Ibrahim, H. R., & Kato, Y. (1999). Improvement of heat stability and emulsifying activity of ovalbumin by conjugation with glucuronic acid through the Maillard reaction. Food Research International, 32, 129–133. Bals, A., & Kulozik, U. (2003). Effect of pre-heating on the foaming properties of whey protein isolate using a membrane foaming apparatus. International Dairy Journal, 13, 903–908. Buchberger, J., & Dovc, P. (2000). Lactoprotein genetic variants in cattle. Food Technology and Biotechnology, 38, 91–98. Campbell, G. M., & Mougeot, E. (1999). Creation and characterisation of aerated food products. Trends in Food Science and Technology, 10, 283–296. Davis, J. P., & Foegeding, E. A. (2004). Foaming and interfacial properties of polymerized whey protein isolate. Journal of Food Science, 69, 404–410. Davis, J. P., & Foegeding, E. A. (2007). Comparisons of the foaming and interfacial properties of whey protein isolate and egg white proteins. Colloids and Surfaces B: Biointerfaces, 54, 200–210. Derjaguin, B. V. (1989). Theory of stability of colloids and thin films. New York, NY, USA: Consultants Bureau. Dickinson, E. (1999). Adsorbed protein layers at fluid interfaces: interactions, structure and surface rheology. Colloids and Surfaces B: Biointerfaces, 15, 161–176. Foegeding, E. A., Luck, P. J., & Davis, J. P. (2006). Factors determining the physical properties of protein foams. Food Hydrocolloids, 20, 284–292. Gonzalez-Ramos, D., & Gonzalez, R. (2006). Genetic determinants of the release of mannoproteins of enological interest by Saccharomyces cerevisiae. Journal of Agricultural and Food Chemistry, 54, 9411–9416. Holland, J. W., Deeth, H. C., & Alewood, P. F. (2006). Resolution and characterisation of multiple isoforms of bovine kappa-casein by 2-DE following a reversible cysteine-tagging enrichment strategy. Proteomics, 6, 3087–3095. Kato, A., Murata, K., & Kobayashi, K. (1988). Preparation and characterization of ovalbumin-dextran conjugate having excellent emulsifying properties. Journal of Agricultural and Food Chemistry, 36, 421–425.
Kreuß, M., Krause, I., & Kulozik, U. (2008). Separation of a glycosylated and nonglycosylated fraction of caseinomacropeptide using different anion-exchange stationary phases. Journal of Chromatography A, 1208, 126–132. Lemeste, M., Colas, B., Simatos, D., Closs, B., Courthaudon, J. L., & Lorient, D. (1990). Contribution of protein flexibility to the foaming properties of casein. Journal of Food Science, 55, 1445–1447. Marshall, S. C. (1991). Casein macropeptide from whey – a new product opportunity. Food Research Quarterly, 51, 86–91. Martin, A. H., Grolle, K., Bos, M. A., Cohen-Stuart, M. A., & van Vliet, T. (2002). Network forming properties of various proteins adsorbed at the air/water interface in relation to foam stability. Journal of Colloid and Interface Science, 254, 173–178. Murray, B. S. (2007). Stabilization of bubbles and foams. Current Opinion in Colloid and Interface Science, 12, 232–241. Nicorescu, I., Loisel, C., Vial, C., Riaublanc, A., Djelveh, G., Cuvelier, G., et al. (2008). Combined effect of dynamic heat treatment and ionic strength on the properties of whey protein foams – part II. Food Research International, 41, 980–988. Nunez, Y. P., Carrascosa, A. V., Gonzalez, R., Polo, M. C., & Martinez-Rodriguez, A. (2006). Isolation and characterization of a thermally extracted yeast cell wall fraction potentially useful for improving the foaming properties of sparkling wines. Journal of Agricultural and Food Chemistry, 54, 7898–7903. Overbeek, J. Th. G. (1977). Recent developments in the understanding of colloid stability. In M. Kerker, A. C. Zettlemoyer, & R. L. Rowell (Eds.), Colloid and interface science (pp. 431–445). New York, NY, USA: Academic Press. Raikos, V., Campbell, L., & Euston, S. R. (2007). Effects of sucrose and sodium chloride on foaming properties of egg white proteins. Food Research International, 40, 347–355. Rodriguez Patino, J. M., Carrera Sanchez, C., & Rodriguez Nino, M. R. (2008). Implications of interfacial characteristics of food foaming agents in foam formulations. Advances in Colloid and Interface Science, 140, 95–113. Rullier, B., Novales, B., & Axelos, M. A. V. (2008). Effect of protein aggregates on foaming properties of b-lactoglobulin. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 330, 96–102. Thoma¨-Worringer, C., Sorensen, J., & Lopez-Fandino, R. (2006). Health effects and technological features of caseinomacropeptide. International Dairy Journal, 16, 1324–1333. Uversky, V. N. (2002). Natively unfolded proteins: a point where biology waits for physics. Protein Science, 11, 739–756. Wilde, P. J., & Clark, D. C. (1996). Foam formation and stability. InMethods of testing protein functionality, Vol 1 (pp. 110–152). London, UK: Blackie Academic. Zhang, Z., Dalgleish, D. G., & Goff, H. D. (2004). Effect of pH and ionic strength on competitive protein adsorption to air/water interfaces in aqueous foams made with mixed milk proteins. Colloids and Surfaces B: Biointerfaces, 34, 113–121. Zhang, Z., & Goff, H. D. (2004). Protein distribution at air interfaces in dairy foams and ice cream as affected by casein dissociation and emulsifiers. International Dairy Journal, 14, 647–657.