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Proteomic profiling of the coagulation of milk proteins induced by glucono-delta-lactone
Food Hydrocolloids 52 (2016) 137e143
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Proteomic profiling of the coagulation of milk proteins induced by glucono-delta-lactone Ying-Ching Chen a, Chun-Chi Chen a, b, Shui-Tein Chen c, Jung-Feng Hsieh a, b, * a
Department of Food Science, Fu Jen Catholic University, Taipei 242, Taiwan Ph.D. Program in Nutrition & Food Science, Fu Jen Catholic University, Taipei 242, Taiwan c Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2015 Received in revised form 13 May 2015 Accepted 9 June 2015 Available online 20 June 2015
This study investigated the glucono-delta-lactone (GDL)-induced coagulation of milk proteins at 30 C. The addition of 0.5 M GDL caused milk proteins to coagulate following a 1 h incubation period. Approximately 90.7% of milk proteins were coagulated into the milk pellet fraction (MPF), and the protein concentration of the milk supernatant fraction (MSF) decreased from 29.2 ± 1.1 mg mL1 (control) to 2.7 ± 1.1 mg mL1. The SDS-PAGE analysis demonstrated that the protein bands corresponding to aS-casein, b-casein and k-casein in the MSF decreased to 0.2 ± 0.1, 0.5 ± 0.2 and 0.5 ± 0.3% of their original levels, respectively. However, only 29.5% of the b-lactoglobulin was coagulated into the MPF following the treatment with 0.5 M GDL. Two-dimensional electrophoresis analysis indicated that isomers of aS1-casein, aS2-casein, b-casein and k-casein, as well as a fraction of b-lactoglobulin and alactalbumin, were coagulated from the MSF into the MPF following incubation with 0.5 M GDL. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Milk protein Glucono-delta-lactone Proteomics Coagulation
1. Introduction Milk, a colloidal solution containing lipids, lactose and approximately 3% protein. Milk proteins are fundamental functional constitutes for food manufacturing because of their high nutritional benefits and unique structural and physicochemical properties (Ye & Harte, 2013). Whey proteins, which are composed primarily of blactoglobulin (b-LG) and a-lactalbumin (a-LA), are major milk proteins and account for 20% of total milk protein. The isoelectric points (pI) of b-LG and a-LA are 5.2 and 4.8, respectively (Haginaka, 2000). The other major family of milk proteins is casein, which accounts for 80% of the total milk protein (Morris, Foster, & Harding, 2000). The casein micelles consist of a complex mixture of four common caseins, aS1-casein (aS1-CN), aS2-casein (aS2-CN), bcasein (b-CN) and k-casein (k-CN). The pI of these proteins range from 4.9 to 5.0 for aS1-CN, from 5.2 to 5.4 for aS2-CN, from 5.1 to 5.4 for b-CN, and from 5.4 to 5.6 for k-CN (Wal, 2002). Casein micelles can be processed into a wide variety of dairy products, such as cheese and yogurt. The coagulation of milk proteins in the cheesemaking process also leads to the destabilization of casein micelles
* Corresponding author. 510 Zhongzheng Road, Xinzhuang District, New Taipei City 24205, Taiwan. Tel.: þ886 2 29052516; fax: þ886 2 29053622. E-mail address: [email protected] (J.-F. Hsieh). http://dx.doi.org/10.1016/j.foodhyd.2015.06.005 0268-005X/© 2015 Elsevier Ltd. All rights reserved.
(Singh, Roberts, Munro, & Teo, 1996). In brief, the acidic coagulation of casein micelles commonly involves the fermentation of milk using lactic acid bacteria to produce acid-curd cheese; this is inturn achieved through the conversion of lactose to lactic acid. An alternative approach achieves acidification using an acidulant, such as glucono-delta-lactone (GDL) (Guinee, Feeney, Auty, & Fox, 2002). Fetahagi c, Macej, Djurdjevic, and Jovanovi c (2002) reported that milk acidified under 0.5e3.0% (w/w) GDL at 25e45 C during cheese preparation. The use of GDL helps reduce a number of difficulties associated with the use of starter lactic bacteria, such as variations related to culture type. Furthermore, GDL does not lead to lactose hydrolysis in milk (Braga, Menossi, & Cunha, 2006). GDL is a carbohydrate that contains a lactone group. GDL hydrolyzes gradually in water to form gluconic acid, causing a reduction in pH (Lucey, Tamehana, Singh, & Munro, 1998). As the pH of milk is decreased from its natural value of 6.7 by the hydrolysis of GDL, the micellar inorganic calcium phosphate gradually dissolves and becomes fully soluble at a pH of approximately 5.2 (Dalgleish & Law, 1989). However, there is only a slight dissociation of casein from the micelle if the acidification is performed at temperatures greater than 25 C (Law & Leaver, 1998). As the pH decreases, the surface charges of the casein micelles are balanced, resulting in the collapse of the k-casein hairy layer. Hence, the steric and electrostatic stabilization are diminished, and at a pH of
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approximately 4.9, the casein micelles coagulate to form a gel (de Kruif, 1997). Therefore, the acidification of milk results in several structural and compositional changes in the casein micelles, which lead to their aggregation at pH 4.9. The addition of GDL promotes the homogeneous acidification of the system. Indeed, in the dairy industry, GDL is used to produce yogurts, cottage cheese and feta cheese because it gives excellent control and reproducibility of the decrease in pH (Martin et al., 2009). At present, proteomic approaches are commonly used to identify milk proteins using two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry. 2-DE is commonly employed for proteomic analyses because of the high-resolution and because of the capability for the simultaneous detection and quantification of thousands of protein spots in the same gel. The proteins in the spots isolated from 2-DE gels are digested with trypsin to yield a large collection of peptides. The complex peptide mixture is then analyzed by matrix-assisted laser-desorption ionization time-offlight (MALDI-TOF) mass spectrometry (Agrawal, Yonekura, Iwahashi, Iwahashi, & Rakwal, 2005). The tandem mass spectra are searched against a protein database to identify the proteins (Hsieh, Yu, Chang, Chen, & Tsai, 2014). In our previously study, we employed 2-DE coupled with mass spectrometry to detect and quantify individual milk proteins (Hsieh & Pan, 2012). The induced gelation of milk proteins by GDL is of considerable importance in the processing of acidified milk products (Takeuchi, Rosiane, & Cunha, 2008). The use of GDL for the coagulation of casein micelles has previously been reported; however, no previous studies have conducted a proteomic analysis to investigate the coagulation of individual milk proteins. Therefore, our use of proteomic analysis (i.e. by way of SDS-PAGE and 2-DE) to study the effects of GDL on the coagulation of individual caseins and whey proteins is a novel contribution. The objective of this study was to analyze the GDL-induced coagulation of milk proteins using a proteomics-based approach. 2. Material and methods 2.1. Preparation of milk samples containing various concentrations of GDL Fresh raw milk from a healthy Holstein-Friesian cow was obtained from a local farm in Taipei in northern Taiwan. The milk was skimmed at 5000 g for 20 min, and the skim milk was subsequently pasteurized at 63 C for 30 min, according to Oeffner et al. (2013). The whey proteins in milk underwent less than 10% denaturation under these pasteurization conditions. The skim milk (30.2 mg mL1) was collected and stored at 4 C. The GDL was obtained from Sigma Chemical Co. (St. Louis, MO, USA). To investigate the effects of GDL on the coagulation of milk proteins, milk samples with varying amounts of GDL (0, 0.1, 0.2, 0.3, 0.4 or 0.5 M) were incubated at 30 C for 1 h. After incubation, the milk samples were fractionated into the milk supernatant fraction (MSF) and the milk pellet fraction (MPF) by centrifugation for 20 min (5000 g). MSF samples (1 mL) were collected, and MPF samples were resuspended in an equal volume (1 mL) of lysis solution containing 7 M urea, 2 M thiourea and 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate prior to use. 2.2. Determinations of protein concentrations and pH values The protein concentrations of the milk samples were determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). The Bio-Rad protein-assay dye was diluted with 4 volumes of water and then mixed with individual standards or milk samples. The absorbance at 595 nm was measured using a VersaMax™ microplate
reader (Molecular Devices Corporation, Sunnyvale, CA, USA), and bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA) was analyzed as the standard. The pH value was measured with a pH meter (Sartorius Basic Meter PB-10, Germany). 2.3. Sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) Milk samples with or without GDL were analyzed by SDS-PAGE. Milk samples were analyzed using a 12.5% separating gel and a 5% stacking gel. For each sample, a 0.1 mL volume of sample was mixed with 0.9 mL of buffer (2% SDS, 5% b-mercaptoethanol, 10% glycerol, 0.02% bromophenol blue, and 70 mM TriseHCl, pH 6.8) and heated to 95 C for 5 min. The samples (6 mL) and a protein ladder were loaded into separate wells. Following electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250. The stained gels were digitized using an EPSON perfection 1270 image scanner (Epson America Inc., Long Beach, CA, USA) and analyzed using the Gel-Pro Analyzer (version 4.0, Media Cybernetics, Inc.) software programs. The level of protein coagulation induced by GDL was evaluated by the magnitudes of the changes in the electrophoretic profiles. 2.4. Two-dimensional electrophoresis (2-DE) Milk samples were analyzed by 2-DE according to the method of Hsieh and Pan (2012). For the first separation, 100 mg of total milk protein was immobilized and loaded onto a pH-gradient (IPG) gel strip (pH 4e7, 18 cm; GE Healthcare) that had been rehydrated for 12 h in a solution containing 7 M urea, 2 M thiourea, 4% 3-[(3cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 40 mM Tris-base, 2% IPG ampholyte, 65 mM 1,4-dithioerythritol (DTE), and 0.0002% bromophenol blue. The isoelectric focusing of the strips was performed at 20 C and 6000 V for a total of 60 kVh using the IPGphor 3 IEF system (GE Healthcare). The strips were equilibrated for 15 min in an equilibration solution (50 mM TriseHCl, pH 8.8, 6 M urea, 2% SDS, 30% glycerol, and 2% DTE) and loaded into the top of a vertical 12.5% SDS-PAGE gel with 0.5% agarose. The second electrophoresis step was performed using a Protean II xi Cell System (Bio-Rad) at 10 mA per gel for 1 h, followed by 45 mA per gel for 5 h until the bromophenol blue traveled to the bottom of the gel. After electrophoresis, the gels were immersed in 10% methanol and 7% acetic acid for 30 min before staining overnight in 350 mL of Sypro® Ruby protein gel-stain solution (Lopez et al., 2000). The developed gels were digitally scanned as 2-D images using a Typhoon 9200 imaging system (Amersham Pharmacia Biotech) and analyzed using the Samespots software program (TotalLab Ltd., Newcastle-upon-Tyne, UK). 2.5. Statistical analysis Data are expressed as the mean values ± standard deviations. The data were analyzed using the Statistical Package for the Social Sciences software program (SPSS for Windows, version 10.0.7C, SPSS Inc., Chicago, IL, USA). The statistical significance of a difference among treatments was determined by one-way ANOVA, followed by a Duncan's multiple-range test to identify the significant differences among means. For the statistical analysis, there were three determinations for each treatment, and the significance level was set at P < 0.05. 3. Results and discussion 3.1. Effect of GDL concentration on the coagulation of milk proteins Milk samples were incubated with varying concentrations of
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GDL (0, 0.1, 0.2, 0.3, 0.4 or 0.5 M) at 30 C for 1 h, and the pH value of the milk supernatant fraction (MSF) was determined. As shown in Fig. 1, the pH values of the MSF samples decreased significantly with an increase in the addition of GDL. The pH value of MSF samples treated with 0, 0.1, 0.2, 0.3, 0.4 and 0.5 M GDL were 6.7, 6.1, 5.7, 5.3, 4.9 and 4.5, respectively. Furthermore, the concentrations of total protein in the MSF and the MPF from treatments with varying concentrations of GDL were also determined. The total protein levels in the MSF and MPF without any GDL treatment were 29.2 ± 1.1 and 0.8 ± 0.8 mg mL1, respectively, indicating that protein coagulation did not occur in milk samples without the addition of GDL. Furthermore, the total protein concentration in the MSF decreased with an increase in the GDL concentration. Approximately 90.7% of the milk proteins were coagulated by 0.5 M GDL, and the total protein concentration in these MSF samples was decreased from 29.2 ± 1.1 mg mL1 (control) to 2.7 ± 1.1 mg mL1. The concentration of total protein in the MPF was obviously increased by the addition of 0.5 M GDL (P < 0.05). Martin et al. (2009) reported that the hydrolysis of GDL into gluconic acid results in a reduction in milk pH. As the pH decreases, micellar phenomena occur during the entire acidification phase that precedes milk gelation: first, the micellar hairy layer collapses; then, the destabilized casein particles aggregate together. Finally, the milk gelation is formed (Banon & Hardy, 1992). Once the pH of the milk (pH 6.7) decreases to 4.7e5.0, the calcium-phosphate linkages between casein submicelles are destabilized, leading to hydrophobic associations and the subsequent precipitation of casein (Xiong, 2009). As previously stated, the addition of GDL to milk results in a reduction in milk pH. Therefore, our results demonstrate that the addition of 0.5 M GDL (pH 4.55) indirectly causes milk proteins to coagulate via the direct acidification of the milk. 3.2. SDS-PAGE analysis of the effect of GDL concentration on casein and whey proteins Milk samples were incubated with various concentrations of GDL (0, 0.1, 0.2, 0.3, 0.4 or 0.5 M) at 30 C for 1 h and then analyzed by SDS-PAGE. As shown in Fig. 2, SDS-PAGE separated the major milk proteins, including the as-CN, b-CN, k-CN and b-LG in milk. The molecular weight of each protein was 23.6, 25.2, 24.0 and 19.0 kDa, respectively (Wal, 2002). The fraction corresponding to a molecular weight of 26 kDa contained as-CN, including aS1-CN and aS2-CN. Thus, as the pH decreases, GDL can coagulate milk proteins when the pH is similar to or below the theoretical pI values for the milk proteins because the net charge of milk proteins is almost zero,
Fig. 1. Changes in the total protein content and pH of milk samples treated with various concentrations of GDL (0, 0.1, 0.2, 0.3, 0.4 or 0.5 M). MSF: milk supernatant fraction; MPF: milk pellet fraction.
Fig. 2. Changes in the SDS-PAGE profiles of milk samples treated with various concentrations of GDL (0, 0.1, 0.2, 0.3, 0.4 or 0.5 M) incubated at 30 C for 1 h. A: milk supernatant fraction (MSF); B: milk pellet fraction (MPF); M: protein marker.
leading to precipitation. The concentrations of the as-CN, b-CN and k-CN in the MSF decreased with an increase in the concentration of GDL (Fig. 2A). Only a portion of the as-CN and b-CN in the MSF were coagulated by the treatments of 0.2 and 0.3 M GDL, but these proteins were apparent in the MPF (Fig. 2B). In contrast, the vast majority of the as-CN, b-CN and k-CN in the MSF were coagulated by the additions of 0.4 and 0.5 M GDL. As previously mentioned, the pH values of MSF samples treated with 0.2, 0.3, 0.4 and 0.5 M GDL were 5.7, 5.3, 4.9 and 4.5, respectively. Euston, Finnigan, and Hirst (2002) have suggested that caseins are easily precipitated by lowering the pH to their approximate pI (pH 4.6). Donato, Alexander, and Dalgleish (2007) reported that the acidification of milk results in several structural and compositional changes in casein micelles that result in their aggregation at pH 4.9. Therefore, the aS-CN, b-CN and k-CN were almost completely removed from the MSF by the additions of 0.4 and 0.5 M GDL. Hence, our results demonstrate that the addition of 0.5 M GDL to milk induces the destabilization and coagulation of the casein micelles. These precipitated casein micelles include as-CN, b-CN and k-CN. However, we found that, although the pI of b-LG is 5.2, only a portion of the b-LG was depleted by the additions of 0.4 and 0.5 M GDL, indicating that b-LG remains in solution at its pI. Labropoulos, Palmer, and Lopez (1981) reported that the pasteurization of milk at 63 C for 30 min resulted in less than 10% denaturation of whey proteins, including b-LG. This is a clear indication that heating milk proteins causes a portion of the b-LG to unfold, which cancels the hydrophilic, negatively charged nature of the surface. As previously
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Fig. 3. Two-dimensional gel electrophoresis analysis of milk proteins. Milk proteins were separated by SDS-PAGE on a 12.5% gel using a pH 4e7 IPG strip. The arrows indicate protein spots that were analyzed in this study. MW: molecular weight; pI: isoelectric points. Spots 1 and 2 were isomers of aS1-CN. Spots 3 to 5 were isomers of aS2-CN. Spots 6 and 7 were isomers of b-CN. Spots 8 to 11 were isomers of k-CN. Spots 12 and 13 were isomers of b-LG, and spot 14 was a-LA.
mentioned, most casein micelles as well as a portion of the b-LG were depleted by the addition of 0.4 and 0.5 M GDL. Thus, denatured b-LG appears to have been co-precipitated or entrapped within the curd. Wong, Camirand, and Pavlath (1996) suggested that the resistance of b-LG to denaturation, even at a pH of 2.0, demonstrates that it is remarkably acid-stable. Thus, only a small fraction of the b-LG was depleted in solution at its pI. Densitograms corresponding to the SDS-PAGE analyses of the milk samples treated with various concentrations of GDL were analyzed. With the addition of 0.4 M GDL, the total intensities of asCN, b-CN, k-CN and b-LG in the MSF decreased to 40.0 ± 4.1, 34.1 ± 5.7, 20.0 ± 4.6 and 88.9 ± 4.1% of the control values, respectively. After the addition of 0.5 M GDL, the total intensities of as-CN, b-CN, k-CN and b-LG in the MSF decreased to 0.2 ± 0.1, 0.2 ± 0.1, 0.6 ± 0.1 and 70.5 ± 2.4% of the controls, respectively. In addition, the intensities of aS-CN, b-CN, k-CN and b-LG (to a lower extent) increased markedly in the MPF following the addition of 0.5 M GDL. Lucey and Singh (2002) reported that, during milk acidification, casein particles aggregate as a result of (mainly) charge neutralization, which results in the formation of chains and clusters that are linked together to form a three-dimensional network. As the pH decreases, GDL can coagulate milk proteins when the pH is similar to or below the theoretical pI values of milk proteins because the net charge of milk proteins is close to zero, resulting in a precipitation phase. Euston et al. (2002) have suggested that caseins, which are disordered proteins, are insensitive to heating but are easily precipitated by lowering the pH to their pI (approximately pH 4.6). In contrast, whey proteins remain in solution at their pI. Correspondingly, our results demonstrate that the
Fig. 4. Changes in the two-dimensional polyacrylamide gel electrophoresis profiles of milk proteins following treatment with various concentrations of GDL (0, 0.3, 0.4 or 0.5 M) at 30 C for 1 h. MSF: milk supernatant fraction. MW: molecular weight; pI: isoelectric points.
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Fig. 5. Changes in the two-dimensional polyacrylamide gel electrophoresis profiles of milk proteins following treatment with various concentrations of GDL (0, 0.3, 0.4 or 0.5 M) at 30 C for 1 h. MPF: milk pellet fraction. MW: molecular weight; pI: isoelectric points.
addition of 0.5 M GDL (pH 4.6) causes aS1-CN, aS2-CN, b-CN, k-CN and a portion of b-LG to aggregate together. 3.3. 2-DE analysis of the effect of GDL concentration on caseins and whey proteins The 2-DE image of MSF without any added GDL is shown in Fig. 3. In total, 14 proteins were selected from the 2-DE gel and assigned individual numbers. These milk proteins were identified as casein and whey proteins by direct comparison with our previous studies (Hsieh & Pan, 2012). These proteins grouped into isomers of aS1-CN, aS2-CN, b-CN, k-CN, b-LG, and a-LA. Spots 1 and 2 were isomers of aS1-CN. Spots 3 to 5 were isomers of aS2-CN. Spots 6 and 7 were isomers of b-CN. Spots 8 to 11 were isomers of k-CN. Spots 12 and 13 were isomers of b-LG, and spot 14 was a-LA. The aS1-CN, aS2-CN, b-CN, and k-CN proteins are known to be incorporated into casein micelles (Müller-Buschbaum, Gebhardt, Roth, Metwalli, & Doster, 2007). Livney and Dalgleish (2004) reported that k-CN is a calcium-insensitive glycoprotein, of an extremely amphiphilic nature, with a hydrophobic N-terminal portion and a hydrophilic glycosylated C-terminal portion. Barros, Ferreira, Silva, and Malcata (2001) reported that b-LG and a-LA are major components of whey protein isolates. The addition of 0.5 M GDL efficiently coagulated milk proteins into the MPF. Therefore, milk samples were incubated with varying concentrations of GDL (0, 0.3, 0.4 or 0.5 M) at 30 C for 1 h, and the MSF samples were electrophoretically separated on 2D gels. Based on the 2-DE results, decreases in aS1-CN, aS2-CN, b-CN, k-CN, b-LG, and a-LA levels were observed in the MSF with the addition of GDL for a 1-h incubation
period, indicating that aS1-CN, aS2-CN, b-CN, k-CN, b-LG, and a-LA coagulation did occur under these conditions (Fig. 4). We observed that only a portion of aS1-CN, aS2-CN, b-CN, k-CN, b-LG and a-LA in the MSF were coagulated by the addition of 0.4 M GDL. After treatment with 0.5 M GDL, the caseins were almost completely removed from the MSF, including aS1-CN, aS2-CN, b-CN and k-CN, into the MPF (Fig. 5). The appearance of individual aS1-CN, aS2-CN,
Fig. 6. Relative abundances of milk protein spots after treatment with various concentrations of GDL (0, 0.3, 0.4 or 0.5 M) at 30 C for 1 h. MSF: milk supernatant fraction. The histograms indicate the fold-change in the protein spots, which were determined using the Samespots software program. Vertical bars represent standard deviations.
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Table 1 Coagulation of as1-CN, as2-CN, b-CN and k-CN in the MSF following the addition of 0.5 M GDL. Spot no.a
Protein name
Isoelectric points (pI)
Remaining proportions (fold)
1 2 3 4 5 6 7 8 9 10 11
as1-CN as1-CN as2-CN as2-CN as2-CN b-CN b-CN k-CN k-CN k-CN k-CN
4.9 4.9 4.9e5.0 4.9e5.0 4.9e5.0 5.2e5.4 5.2e5.4 5.1e5.4 5.1e5.4 5.1e5.4 5.1e5.4
0.01 0.01 0.01 0.01 0.02 0.01 0.03 0.03 0.02 0.02 0.02
a
Spot no. ¼ spot number, corresponds to Fig. 3.
b-CN and k-CN molecules in the MPF is evidence for the coagulation of protein by GDL. However, only a portion of the b-LG and a-LA were removed by the addition of 0.5 M GDL, indicating that b-LG and a-LA remain in solution at their pI. Anema, Lowe, and Lee (2004) reported that, in milk, the native whey proteins are soluble at their pI. Furthermore, the strong cation binding to the calcium site increases the stability of a-LA (Permyakov & Berliner, 2000). In addition, the preponderance of non-ionizable polar side chain groups in whey proteins renders them soluble even at their isoelectric pH (Xiong, 2009). Densitograms corresponding to the 2-DE images of milk samples from treatments with various concentrations of GDL (0, 0.3, 0.4 or 0.5 M) were also generated, and the relative changes in individual milk protein levels, compared to the control, are shown in Fig. 6. As previously noted, the majority of the aS1-CN, aS2-CN, b-CN and k-CN in the MSF were coagulated by the addition of 0.5 M GDL. The remaining proportions of aS1-CN (spot 1), aS1-CN (spot 2), aS2CN (spot 3), aS2-CN (spot 4), aS2-CN (spot 5), b-CN (spot 6), b-CN (spot 7), k-CN (spot 8), k-CN (spot 9), k-CN (spot 10) and k-CN (spot 11) in the GDL-treated MSF were 0.01, 0.01, 0.01, 0.01, 0.02, 0.01, 0.03, 0.03, 0.02, 0.02 and 0.02, respectively (Table 1). We observed
that only a portion of the b-LG and a-LA in the MSF were also coagulated by the addition of 0.5 M GDL. The remaining proportions of b-LG (spot 12), b-LG (spot 13) and a-LA (spot 14) in the GDL-treated MSF were 0.77, 0.73 and 0.64, respectively (Fig. 6). As previously mentioned, the addition of 0.5 M GDL to milk induces the destabilization and coagulation of casein micelles, and the isomers of aS1-CN, aS2-CN, b-CN, and k-CN, and a fraction of the bLC and a-LA, in the milk are coagulated into the pellet. The complexation between the GDL and the milk proteins yields insoluble complexes, resulting in phase separation. 3.4. Potential mechanism for the effect of GDL concentration on milk proteins Dickinson (2006) noted that the acidification process converts the liquid-like dispersion of casein particles and whey proteins into a soft, solid-like aggregated network. Our results clearly demonstrate that the total protein in the MPF increased with the addition of 0.5 M GDL, indicating that GDL reacts with caseins and a portion of whey proteins during milk coagulation. Wal (2002) reported that approximately 80% of milk protein is composed of casein, and 20% is composed of whey proteins. The results indicate that the coagulated proteins consist of the entire 80% attributed to casein and 14.3% of the total 20% of whey proteins. Based on the abovedescribed results, a reaction scheme for the effect of GDL on the coagulation of individual milk proteins is depicted in Fig. 7. The GDL-mediated coagulation reaction has been described as a twostep process. As previously mentioned, the hydrolysis of GDL to gluconic acid results in a decrease in the milk pH. The pI of aS1-CN, aS2-CN, b-CN, k-CN, b-LG and a-LA are 4.9, 4.9e5, 5.2e5.4, 5.1e5.4, 5.4e5.6, 5.2 and 4.8, respectively. The first step of the reaction scheme is a process in which GDL catalyzes the acidification of the milk. As the pH decreases, micellar phenomena occur during the entire acidification phase that precedes milk coagulation. The micellar hairy layer (k-CN) collapses, and the destabilized casein particles then aggregate together. The second step involves the GDL-mediated coagulation of the aS1-CN, aS2-CN, b-CN, and k-CN, as well as a portion of the b-LC and a-LA. However, the degree of coagulation of the whey proteins was less than that of the caseins.
Fig. 7. Reaction scheme for the effect of GDL on the coagulation of milk proteins.
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The coagulated milk proteins contained aS1-CN, aS2-CN, b-CN, k-CN, b-LC and a-LA. 4. Conclusions A proteomic analysis of the effect of GDL concentration on the coagulation of individual milk proteins was performed. The results presented in this study clearly demonstrate that the addition of GDL to milk induces the destabilization and coagulation of casein micelles. These casein micelles contain aS1-CN, aS2-CN, b-CN and kCN. The SDS-PAGE and 2-DE analyses revealed that the aS1-CN, aS2CN, b-CN, and k-CN, as well as a portion of the b-LG and a-LA, were coagulated by the 0.5 M GDL treatment. Based on our results, proteomic approaches can be successfully employed to analyze the effects of GDL on individual milk proteins. Acknowledgments We thank the National Science Council in Taiwan for grant support (NSC 102-2313-B-030-001). References Agrawal, G. K., Yonekura, M., Iwahashi, Y., Iwahashi, H., & Rakwal, R. (2005). System, trends and perspectives of proteomics in dicot plants Part I: technologies in proteome establishment. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences, 815, 109e123. Anema, S. G., Lowe, E. K., & Lee, S. K. (2004). Effect of pH at heating on the acidinduced aggregation of casein micelles in reconstituted skim milk. LWT e Food Science and Technology, 37, 779e787. Banon, S., & Hardy, J. (1992). A colloidal approach of milk acidification by gluconodelta-lactone. Journal of Dairy Science, 75, 935e941. Barros, R. M., Ferreira, C. A., Silva, S. V., & Malcata, F. X. (2001). Quantitative studies on the enzymatic hydrolysis of milk proteins brought about by cardosins precipitated by ammonium sulfate. Enzyme and Microbial Technology, 29, 541e547. Braga, A. L. M., Menossi, M., & Cunha, R. L. (2006). The effect of the glucono-dlactone/caseinate ratio on sodium caseinate gelation. International Dairy Journal, 16, 389e398. Dalgleish, D. G., & Law, A. J. R. (1989). pH-induced dissociation of bovine casein micelles. II. Mineral solubilization and its relation to casein release. Journal of Dairy Research, 56, 727e735. Dickinson, E. (2006). Structure formation in casein-based gels, foams, and emulsions. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 288, 3e11. Donato, L., Alexander, M., & Dalgleish, D. G. (2007). Acid gelation in heated and unheated milks: interactions between serum protein complexes and the surfaces of casein micelles. Journal of Agricultural and Food Chemistry, 55, 4160e4168. Euston, S. R., Finnigan, S. R., & Hirst, R. L. (2002). Kinetics of droplet aggregation in heated whey protein-stabilized emulsions: effect of polysaccharides. Food Hydrocolloids, 16, 499e505. Fetahagi c, S., Ma cej, O., Djurdjevi c, J. D., & Jovanovi c, S. (2002). The influence of GDL concentration on milk pH change during acid coagulation. Journal of Agricultural Science, 47, 75e85. Guinee, T. P., Feeney, E. P., Auty, M. A., & Fox, P. F. (2002). Effect of pH and calcium concentration on some textural and functional properties of mozzarella cheese.
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Journal of Dairy Science, 85, 1655e1669. Haginaka, J. (2000). Enantiomer separation of drugs by capillary electrophoresis using proteins as chiral selectors. Journal of Chromatography A, 875, 235e254. Hsieh, J. F., & Pan, P. H. (2012). Proteomic profiling of the coagulation of milk proteins induced by chymosin. Journal of Agricultural and Food Chemistry, 60, 2039e2045. Hsieh, J. F., Yu, C. J., Chang, J. Y., Chen, S. T., & Tsai, H. Y. (2014). Microbial transglutaminase-induced polymerization of b-conglycinin and glycinin in soymilk: a proteomics approach. Food Hydrocolloids, 35, 678e685. de Kruif, C. G. (1997). Skim milk acidification. Journal of Colloid and Interface Science, 185, 19e25. Labropoulos, A. E., Palmer, J. K., & Lopez, A. (1981). Whey protein denaturation of UHT processed milk and its effect on rheology of yogurt. Journal of Texture Studies, 12, 365e374. Law, A. J. R., & Leaver, J. (1998). Effects of acidification and storage of milk on dissociation of bovine casein micelles. Journal of Agricultural and Food Chemistry, 46, 5008e5016. Livney, Y. D., & Dalgleish, D. G. (2004). Specificity of disulfide bond formation during thermal aggregation in solutions of b-lactoglobulin B and k-casein A. Journal of Agricultural and Food Chemistry, 52, 5527e5532. Lopez, M. F., Berggren, K., Chernokalskaya, E., Lazarev, A., Robinson, M., & Patton, W. F. (2000). A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling. Electrophoresis, 21, 3673e3683. Lucey, J. A., & Singh, H. (2002). Acid coagulation of milk. In P. F. Fox, & L. H. McSweeney (Eds.) (2nd ed.)Proteins (1)Advanced dairy chemistry Gaithersburg: Aspen. Lucey, J. A., Tamehana, M., Singh, H., & Munro, P. A. (1998). A comparison of the formation, rheological properties and microstructure of acid skim milk gels made with a bacterial culture or glucono-d-lactone. Food Research International, 31, 147e155. Martin, F., Cayot, N., Marin, A., Journaux, L., Cayot, P., Gervais, P., et al. (2009). Effect of oxidoreduction potential and of gas bubbling on rheological properties and microstructure of acid skim milk gels acidified with glucono-delta-lactone. Journal of Dairy Science, 92, 5898e5906. Morris, G. A., Foster, T. J., & Harding, S. E. (2000). Further observations on the size, shape, and hydration of casein micelles from novel analytical ultracentrifuge and capillary viscometry approaches. Biomacromolecules, 1, 764e767. Müller-Buschbaum, P., Gebhardt, R., Roth, S. V., Metwalli, E., & Doster, W. (2007). Effect of calcium concentration on the structure of casein micelles in thin films. Biophysical Journal, 93, 960e968. Oeffner, S. P., Qu, Y., Just, J., Quezada, N., Ramsing, E., Keller, M., et al. (2013). Effect of flaxseed supplementation rate and processing on the production, fatty acid profile, and texture of milk, butter, and cheese. Journal of Dairy Science, 96, 1177e1188. Permyakov, E. A., & Berliner, L. J. (2000). a-Lactalbumin: structure and function. FEBS Letters, 473, 269e274. Singh, H., Roberts, M. S., Munro, P. A., & Teo, C. T. (1996). Acid-induced dissociation of casein micelles in milk: effects of heat treatment. Journal of Dairy Science, 79, 1340e1346. Takeuchi, K. P., Rosiane, L., & Cunha, R. L. (2008). Influence of ageing time on sodium caseinate gelation induced by glucono-d-lactone at different temperatures. Dairy Science & Technology, 88, 667e681. Wal, J. M. (2002). Cow's milk proteins/allergens. Annals of Allergy Asthma and Immunology, 89, 3e10. Wong, D. W., Camirand, W. M., & Pavlath, A. E. (1996). Structures and functionalities of milk proteins. Critical Reviews in Food Science and Nutrition, 36, 807e844. (Ed.), Ingredients in meat products. Xiong, X. L. (2009). Dairy proteins. In R. Tarte New York, USA: Springer. Ye, R., & Harte, F. (2013). Casein maps: effect of ethanol, pH, temperature, and CaCl2 on the particle size of reconstituted casein micelles. Journal of Dairy Science, 96, 799e805.
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Proteomic profiling of the coagulation of milk proteins induced by glucono-delta-lactone Ying-Ching Chen a, Chun-Chi Chen a, b, Shui-Tein Chen c, Jung-Feng Hsieh a, b, * a
Department of Food Science, Fu Jen Catholic University, Taipei 242, Taiwan Ph.D. Program in Nutrition & Food Science, Fu Jen Catholic University, Taipei 242, Taiwan c Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2015 Received in revised form 13 May 2015 Accepted 9 June 2015 Available online 20 June 2015
This study investigated the glucono-delta-lactone (GDL)-induced coagulation of milk proteins at 30 C. The addition of 0.5 M GDL caused milk proteins to coagulate following a 1 h incubation period. Approximately 90.7% of milk proteins were coagulated into the milk pellet fraction (MPF), and the protein concentration of the milk supernatant fraction (MSF) decreased from 29.2 ± 1.1 mg mL1 (control) to 2.7 ± 1.1 mg mL1. The SDS-PAGE analysis demonstrated that the protein bands corresponding to aS-casein, b-casein and k-casein in the MSF decreased to 0.2 ± 0.1, 0.5 ± 0.2 and 0.5 ± 0.3% of their original levels, respectively. However, only 29.5% of the b-lactoglobulin was coagulated into the MPF following the treatment with 0.5 M GDL. Two-dimensional electrophoresis analysis indicated that isomers of aS1-casein, aS2-casein, b-casein and k-casein, as well as a fraction of b-lactoglobulin and alactalbumin, were coagulated from the MSF into the MPF following incubation with 0.5 M GDL. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Milk protein Glucono-delta-lactone Proteomics Coagulation
1. Introduction Milk, a colloidal solution containing lipids, lactose and approximately 3% protein. Milk proteins are fundamental functional constitutes for food manufacturing because of their high nutritional benefits and unique structural and physicochemical properties (Ye & Harte, 2013). Whey proteins, which are composed primarily of blactoglobulin (b-LG) and a-lactalbumin (a-LA), are major milk proteins and account for 20% of total milk protein. The isoelectric points (pI) of b-LG and a-LA are 5.2 and 4.8, respectively (Haginaka, 2000). The other major family of milk proteins is casein, which accounts for 80% of the total milk protein (Morris, Foster, & Harding, 2000). The casein micelles consist of a complex mixture of four common caseins, aS1-casein (aS1-CN), aS2-casein (aS2-CN), bcasein (b-CN) and k-casein (k-CN). The pI of these proteins range from 4.9 to 5.0 for aS1-CN, from 5.2 to 5.4 for aS2-CN, from 5.1 to 5.4 for b-CN, and from 5.4 to 5.6 for k-CN (Wal, 2002). Casein micelles can be processed into a wide variety of dairy products, such as cheese and yogurt. The coagulation of milk proteins in the cheesemaking process also leads to the destabilization of casein micelles
* Corresponding author. 510 Zhongzheng Road, Xinzhuang District, New Taipei City 24205, Taiwan. Tel.: þ886 2 29052516; fax: þ886 2 29053622. E-mail address: [email protected] (J.-F. Hsieh). http://dx.doi.org/10.1016/j.foodhyd.2015.06.005 0268-005X/© 2015 Elsevier Ltd. All rights reserved.
(Singh, Roberts, Munro, & Teo, 1996). In brief, the acidic coagulation of casein micelles commonly involves the fermentation of milk using lactic acid bacteria to produce acid-curd cheese; this is inturn achieved through the conversion of lactose to lactic acid. An alternative approach achieves acidification using an acidulant, such as glucono-delta-lactone (GDL) (Guinee, Feeney, Auty, & Fox, 2002). Fetahagi c, Macej, Djurdjevic, and Jovanovi c (2002) reported that milk acidified under 0.5e3.0% (w/w) GDL at 25e45 C during cheese preparation. The use of GDL helps reduce a number of difficulties associated with the use of starter lactic bacteria, such as variations related to culture type. Furthermore, GDL does not lead to lactose hydrolysis in milk (Braga, Menossi, & Cunha, 2006). GDL is a carbohydrate that contains a lactone group. GDL hydrolyzes gradually in water to form gluconic acid, causing a reduction in pH (Lucey, Tamehana, Singh, & Munro, 1998). As the pH of milk is decreased from its natural value of 6.7 by the hydrolysis of GDL, the micellar inorganic calcium phosphate gradually dissolves and becomes fully soluble at a pH of approximately 5.2 (Dalgleish & Law, 1989). However, there is only a slight dissociation of casein from the micelle if the acidification is performed at temperatures greater than 25 C (Law & Leaver, 1998). As the pH decreases, the surface charges of the casein micelles are balanced, resulting in the collapse of the k-casein hairy layer. Hence, the steric and electrostatic stabilization are diminished, and at a pH of
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approximately 4.9, the casein micelles coagulate to form a gel (de Kruif, 1997). Therefore, the acidification of milk results in several structural and compositional changes in the casein micelles, which lead to their aggregation at pH 4.9. The addition of GDL promotes the homogeneous acidification of the system. Indeed, in the dairy industry, GDL is used to produce yogurts, cottage cheese and feta cheese because it gives excellent control and reproducibility of the decrease in pH (Martin et al., 2009). At present, proteomic approaches are commonly used to identify milk proteins using two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry. 2-DE is commonly employed for proteomic analyses because of the high-resolution and because of the capability for the simultaneous detection and quantification of thousands of protein spots in the same gel. The proteins in the spots isolated from 2-DE gels are digested with trypsin to yield a large collection of peptides. The complex peptide mixture is then analyzed by matrix-assisted laser-desorption ionization time-offlight (MALDI-TOF) mass spectrometry (Agrawal, Yonekura, Iwahashi, Iwahashi, & Rakwal, 2005). The tandem mass spectra are searched against a protein database to identify the proteins (Hsieh, Yu, Chang, Chen, & Tsai, 2014). In our previously study, we employed 2-DE coupled with mass spectrometry to detect and quantify individual milk proteins (Hsieh & Pan, 2012). The induced gelation of milk proteins by GDL is of considerable importance in the processing of acidified milk products (Takeuchi, Rosiane, & Cunha, 2008). The use of GDL for the coagulation of casein micelles has previously been reported; however, no previous studies have conducted a proteomic analysis to investigate the coagulation of individual milk proteins. Therefore, our use of proteomic analysis (i.e. by way of SDS-PAGE and 2-DE) to study the effects of GDL on the coagulation of individual caseins and whey proteins is a novel contribution. The objective of this study was to analyze the GDL-induced coagulation of milk proteins using a proteomics-based approach. 2. Material and methods 2.1. Preparation of milk samples containing various concentrations of GDL Fresh raw milk from a healthy Holstein-Friesian cow was obtained from a local farm in Taipei in northern Taiwan. The milk was skimmed at 5000 g for 20 min, and the skim milk was subsequently pasteurized at 63 C for 30 min, according to Oeffner et al. (2013). The whey proteins in milk underwent less than 10% denaturation under these pasteurization conditions. The skim milk (30.2 mg mL1) was collected and stored at 4 C. The GDL was obtained from Sigma Chemical Co. (St. Louis, MO, USA). To investigate the effects of GDL on the coagulation of milk proteins, milk samples with varying amounts of GDL (0, 0.1, 0.2, 0.3, 0.4 or 0.5 M) were incubated at 30 C for 1 h. After incubation, the milk samples were fractionated into the milk supernatant fraction (MSF) and the milk pellet fraction (MPF) by centrifugation for 20 min (5000 g). MSF samples (1 mL) were collected, and MPF samples were resuspended in an equal volume (1 mL) of lysis solution containing 7 M urea, 2 M thiourea and 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate prior to use. 2.2. Determinations of protein concentrations and pH values The protein concentrations of the milk samples were determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). The Bio-Rad protein-assay dye was diluted with 4 volumes of water and then mixed with individual standards or milk samples. The absorbance at 595 nm was measured using a VersaMax™ microplate
reader (Molecular Devices Corporation, Sunnyvale, CA, USA), and bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA) was analyzed as the standard. The pH value was measured with a pH meter (Sartorius Basic Meter PB-10, Germany). 2.3. Sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) Milk samples with or without GDL were analyzed by SDS-PAGE. Milk samples were analyzed using a 12.5% separating gel and a 5% stacking gel. For each sample, a 0.1 mL volume of sample was mixed with 0.9 mL of buffer (2% SDS, 5% b-mercaptoethanol, 10% glycerol, 0.02% bromophenol blue, and 70 mM TriseHCl, pH 6.8) and heated to 95 C for 5 min. The samples (6 mL) and a protein ladder were loaded into separate wells. Following electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250. The stained gels were digitized using an EPSON perfection 1270 image scanner (Epson America Inc., Long Beach, CA, USA) and analyzed using the Gel-Pro Analyzer (version 4.0, Media Cybernetics, Inc.) software programs. The level of protein coagulation induced by GDL was evaluated by the magnitudes of the changes in the electrophoretic profiles. 2.4. Two-dimensional electrophoresis (2-DE) Milk samples were analyzed by 2-DE according to the method of Hsieh and Pan (2012). For the first separation, 100 mg of total milk protein was immobilized and loaded onto a pH-gradient (IPG) gel strip (pH 4e7, 18 cm; GE Healthcare) that had been rehydrated for 12 h in a solution containing 7 M urea, 2 M thiourea, 4% 3-[(3cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 40 mM Tris-base, 2% IPG ampholyte, 65 mM 1,4-dithioerythritol (DTE), and 0.0002% bromophenol blue. The isoelectric focusing of the strips was performed at 20 C and 6000 V for a total of 60 kVh using the IPGphor 3 IEF system (GE Healthcare). The strips were equilibrated for 15 min in an equilibration solution (50 mM TriseHCl, pH 8.8, 6 M urea, 2% SDS, 30% glycerol, and 2% DTE) and loaded into the top of a vertical 12.5% SDS-PAGE gel with 0.5% agarose. The second electrophoresis step was performed using a Protean II xi Cell System (Bio-Rad) at 10 mA per gel for 1 h, followed by 45 mA per gel for 5 h until the bromophenol blue traveled to the bottom of the gel. After electrophoresis, the gels were immersed in 10% methanol and 7% acetic acid for 30 min before staining overnight in 350 mL of Sypro® Ruby protein gel-stain solution (Lopez et al., 2000). The developed gels were digitally scanned as 2-D images using a Typhoon 9200 imaging system (Amersham Pharmacia Biotech) and analyzed using the Samespots software program (TotalLab Ltd., Newcastle-upon-Tyne, UK). 2.5. Statistical analysis Data are expressed as the mean values ± standard deviations. The data were analyzed using the Statistical Package for the Social Sciences software program (SPSS for Windows, version 10.0.7C, SPSS Inc., Chicago, IL, USA). The statistical significance of a difference among treatments was determined by one-way ANOVA, followed by a Duncan's multiple-range test to identify the significant differences among means. For the statistical analysis, there were three determinations for each treatment, and the significance level was set at P < 0.05. 3. Results and discussion 3.1. Effect of GDL concentration on the coagulation of milk proteins Milk samples were incubated with varying concentrations of
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GDL (0, 0.1, 0.2, 0.3, 0.4 or 0.5 M) at 30 C for 1 h, and the pH value of the milk supernatant fraction (MSF) was determined. As shown in Fig. 1, the pH values of the MSF samples decreased significantly with an increase in the addition of GDL. The pH value of MSF samples treated with 0, 0.1, 0.2, 0.3, 0.4 and 0.5 M GDL were 6.7, 6.1, 5.7, 5.3, 4.9 and 4.5, respectively. Furthermore, the concentrations of total protein in the MSF and the MPF from treatments with varying concentrations of GDL were also determined. The total protein levels in the MSF and MPF without any GDL treatment were 29.2 ± 1.1 and 0.8 ± 0.8 mg mL1, respectively, indicating that protein coagulation did not occur in milk samples without the addition of GDL. Furthermore, the total protein concentration in the MSF decreased with an increase in the GDL concentration. Approximately 90.7% of the milk proteins were coagulated by 0.5 M GDL, and the total protein concentration in these MSF samples was decreased from 29.2 ± 1.1 mg mL1 (control) to 2.7 ± 1.1 mg mL1. The concentration of total protein in the MPF was obviously increased by the addition of 0.5 M GDL (P < 0.05). Martin et al. (2009) reported that the hydrolysis of GDL into gluconic acid results in a reduction in milk pH. As the pH decreases, micellar phenomena occur during the entire acidification phase that precedes milk gelation: first, the micellar hairy layer collapses; then, the destabilized casein particles aggregate together. Finally, the milk gelation is formed (Banon & Hardy, 1992). Once the pH of the milk (pH 6.7) decreases to 4.7e5.0, the calcium-phosphate linkages between casein submicelles are destabilized, leading to hydrophobic associations and the subsequent precipitation of casein (Xiong, 2009). As previously stated, the addition of GDL to milk results in a reduction in milk pH. Therefore, our results demonstrate that the addition of 0.5 M GDL (pH 4.55) indirectly causes milk proteins to coagulate via the direct acidification of the milk. 3.2. SDS-PAGE analysis of the effect of GDL concentration on casein and whey proteins Milk samples were incubated with various concentrations of GDL (0, 0.1, 0.2, 0.3, 0.4 or 0.5 M) at 30 C for 1 h and then analyzed by SDS-PAGE. As shown in Fig. 2, SDS-PAGE separated the major milk proteins, including the as-CN, b-CN, k-CN and b-LG in milk. The molecular weight of each protein was 23.6, 25.2, 24.0 and 19.0 kDa, respectively (Wal, 2002). The fraction corresponding to a molecular weight of 26 kDa contained as-CN, including aS1-CN and aS2-CN. Thus, as the pH decreases, GDL can coagulate milk proteins when the pH is similar to or below the theoretical pI values for the milk proteins because the net charge of milk proteins is almost zero,
Fig. 1. Changes in the total protein content and pH of milk samples treated with various concentrations of GDL (0, 0.1, 0.2, 0.3, 0.4 or 0.5 M). MSF: milk supernatant fraction; MPF: milk pellet fraction.
Fig. 2. Changes in the SDS-PAGE profiles of milk samples treated with various concentrations of GDL (0, 0.1, 0.2, 0.3, 0.4 or 0.5 M) incubated at 30 C for 1 h. A: milk supernatant fraction (MSF); B: milk pellet fraction (MPF); M: protein marker.
leading to precipitation. The concentrations of the as-CN, b-CN and k-CN in the MSF decreased with an increase in the concentration of GDL (Fig. 2A). Only a portion of the as-CN and b-CN in the MSF were coagulated by the treatments of 0.2 and 0.3 M GDL, but these proteins were apparent in the MPF (Fig. 2B). In contrast, the vast majority of the as-CN, b-CN and k-CN in the MSF were coagulated by the additions of 0.4 and 0.5 M GDL. As previously mentioned, the pH values of MSF samples treated with 0.2, 0.3, 0.4 and 0.5 M GDL were 5.7, 5.3, 4.9 and 4.5, respectively. Euston, Finnigan, and Hirst (2002) have suggested that caseins are easily precipitated by lowering the pH to their approximate pI (pH 4.6). Donato, Alexander, and Dalgleish (2007) reported that the acidification of milk results in several structural and compositional changes in casein micelles that result in their aggregation at pH 4.9. Therefore, the aS-CN, b-CN and k-CN were almost completely removed from the MSF by the additions of 0.4 and 0.5 M GDL. Hence, our results demonstrate that the addition of 0.5 M GDL to milk induces the destabilization and coagulation of the casein micelles. These precipitated casein micelles include as-CN, b-CN and k-CN. However, we found that, although the pI of b-LG is 5.2, only a portion of the b-LG was depleted by the additions of 0.4 and 0.5 M GDL, indicating that b-LG remains in solution at its pI. Labropoulos, Palmer, and Lopez (1981) reported that the pasteurization of milk at 63 C for 30 min resulted in less than 10% denaturation of whey proteins, including b-LG. This is a clear indication that heating milk proteins causes a portion of the b-LG to unfold, which cancels the hydrophilic, negatively charged nature of the surface. As previously
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Fig. 3. Two-dimensional gel electrophoresis analysis of milk proteins. Milk proteins were separated by SDS-PAGE on a 12.5% gel using a pH 4e7 IPG strip. The arrows indicate protein spots that were analyzed in this study. MW: molecular weight; pI: isoelectric points. Spots 1 and 2 were isomers of aS1-CN. Spots 3 to 5 were isomers of aS2-CN. Spots 6 and 7 were isomers of b-CN. Spots 8 to 11 were isomers of k-CN. Spots 12 and 13 were isomers of b-LG, and spot 14 was a-LA.
mentioned, most casein micelles as well as a portion of the b-LG were depleted by the addition of 0.4 and 0.5 M GDL. Thus, denatured b-LG appears to have been co-precipitated or entrapped within the curd. Wong, Camirand, and Pavlath (1996) suggested that the resistance of b-LG to denaturation, even at a pH of 2.0, demonstrates that it is remarkably acid-stable. Thus, only a small fraction of the b-LG was depleted in solution at its pI. Densitograms corresponding to the SDS-PAGE analyses of the milk samples treated with various concentrations of GDL were analyzed. With the addition of 0.4 M GDL, the total intensities of asCN, b-CN, k-CN and b-LG in the MSF decreased to 40.0 ± 4.1, 34.1 ± 5.7, 20.0 ± 4.6 and 88.9 ± 4.1% of the control values, respectively. After the addition of 0.5 M GDL, the total intensities of as-CN, b-CN, k-CN and b-LG in the MSF decreased to 0.2 ± 0.1, 0.2 ± 0.1, 0.6 ± 0.1 and 70.5 ± 2.4% of the controls, respectively. In addition, the intensities of aS-CN, b-CN, k-CN and b-LG (to a lower extent) increased markedly in the MPF following the addition of 0.5 M GDL. Lucey and Singh (2002) reported that, during milk acidification, casein particles aggregate as a result of (mainly) charge neutralization, which results in the formation of chains and clusters that are linked together to form a three-dimensional network. As the pH decreases, GDL can coagulate milk proteins when the pH is similar to or below the theoretical pI values of milk proteins because the net charge of milk proteins is close to zero, resulting in a precipitation phase. Euston et al. (2002) have suggested that caseins, which are disordered proteins, are insensitive to heating but are easily precipitated by lowering the pH to their pI (approximately pH 4.6). In contrast, whey proteins remain in solution at their pI. Correspondingly, our results demonstrate that the
Fig. 4. Changes in the two-dimensional polyacrylamide gel electrophoresis profiles of milk proteins following treatment with various concentrations of GDL (0, 0.3, 0.4 or 0.5 M) at 30 C for 1 h. MSF: milk supernatant fraction. MW: molecular weight; pI: isoelectric points.
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Fig. 5. Changes in the two-dimensional polyacrylamide gel electrophoresis profiles of milk proteins following treatment with various concentrations of GDL (0, 0.3, 0.4 or 0.5 M) at 30 C for 1 h. MPF: milk pellet fraction. MW: molecular weight; pI: isoelectric points.
addition of 0.5 M GDL (pH 4.6) causes aS1-CN, aS2-CN, b-CN, k-CN and a portion of b-LG to aggregate together. 3.3. 2-DE analysis of the effect of GDL concentration on caseins and whey proteins The 2-DE image of MSF without any added GDL is shown in Fig. 3. In total, 14 proteins were selected from the 2-DE gel and assigned individual numbers. These milk proteins were identified as casein and whey proteins by direct comparison with our previous studies (Hsieh & Pan, 2012). These proteins grouped into isomers of aS1-CN, aS2-CN, b-CN, k-CN, b-LG, and a-LA. Spots 1 and 2 were isomers of aS1-CN. Spots 3 to 5 were isomers of aS2-CN. Spots 6 and 7 were isomers of b-CN. Spots 8 to 11 were isomers of k-CN. Spots 12 and 13 were isomers of b-LG, and spot 14 was a-LA. The aS1-CN, aS2-CN, b-CN, and k-CN proteins are known to be incorporated into casein micelles (Müller-Buschbaum, Gebhardt, Roth, Metwalli, & Doster, 2007). Livney and Dalgleish (2004) reported that k-CN is a calcium-insensitive glycoprotein, of an extremely amphiphilic nature, with a hydrophobic N-terminal portion and a hydrophilic glycosylated C-terminal portion. Barros, Ferreira, Silva, and Malcata (2001) reported that b-LG and a-LA are major components of whey protein isolates. The addition of 0.5 M GDL efficiently coagulated milk proteins into the MPF. Therefore, milk samples were incubated with varying concentrations of GDL (0, 0.3, 0.4 or 0.5 M) at 30 C for 1 h, and the MSF samples were electrophoretically separated on 2D gels. Based on the 2-DE results, decreases in aS1-CN, aS2-CN, b-CN, k-CN, b-LG, and a-LA levels were observed in the MSF with the addition of GDL for a 1-h incubation
period, indicating that aS1-CN, aS2-CN, b-CN, k-CN, b-LG, and a-LA coagulation did occur under these conditions (Fig. 4). We observed that only a portion of aS1-CN, aS2-CN, b-CN, k-CN, b-LG and a-LA in the MSF were coagulated by the addition of 0.4 M GDL. After treatment with 0.5 M GDL, the caseins were almost completely removed from the MSF, including aS1-CN, aS2-CN, b-CN and k-CN, into the MPF (Fig. 5). The appearance of individual aS1-CN, aS2-CN,
Fig. 6. Relative abundances of milk protein spots after treatment with various concentrations of GDL (0, 0.3, 0.4 or 0.5 M) at 30 C for 1 h. MSF: milk supernatant fraction. The histograms indicate the fold-change in the protein spots, which were determined using the Samespots software program. Vertical bars represent standard deviations.
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Table 1 Coagulation of as1-CN, as2-CN, b-CN and k-CN in the MSF following the addition of 0.5 M GDL. Spot no.a
Protein name
Isoelectric points (pI)
Remaining proportions (fold)
1 2 3 4 5 6 7 8 9 10 11
as1-CN as1-CN as2-CN as2-CN as2-CN b-CN b-CN k-CN k-CN k-CN k-CN
4.9 4.9 4.9e5.0 4.9e5.0 4.9e5.0 5.2e5.4 5.2e5.4 5.1e5.4 5.1e5.4 5.1e5.4 5.1e5.4
0.01 0.01 0.01 0.01 0.02 0.01 0.03 0.03 0.02 0.02 0.02
a
Spot no. ¼ spot number, corresponds to Fig. 3.
b-CN and k-CN molecules in the MPF is evidence for the coagulation of protein by GDL. However, only a portion of the b-LG and a-LA were removed by the addition of 0.5 M GDL, indicating that b-LG and a-LA remain in solution at their pI. Anema, Lowe, and Lee (2004) reported that, in milk, the native whey proteins are soluble at their pI. Furthermore, the strong cation binding to the calcium site increases the stability of a-LA (Permyakov & Berliner, 2000). In addition, the preponderance of non-ionizable polar side chain groups in whey proteins renders them soluble even at their isoelectric pH (Xiong, 2009). Densitograms corresponding to the 2-DE images of milk samples from treatments with various concentrations of GDL (0, 0.3, 0.4 or 0.5 M) were also generated, and the relative changes in individual milk protein levels, compared to the control, are shown in Fig. 6. As previously noted, the majority of the aS1-CN, aS2-CN, b-CN and k-CN in the MSF were coagulated by the addition of 0.5 M GDL. The remaining proportions of aS1-CN (spot 1), aS1-CN (spot 2), aS2CN (spot 3), aS2-CN (spot 4), aS2-CN (spot 5), b-CN (spot 6), b-CN (spot 7), k-CN (spot 8), k-CN (spot 9), k-CN (spot 10) and k-CN (spot 11) in the GDL-treated MSF were 0.01, 0.01, 0.01, 0.01, 0.02, 0.01, 0.03, 0.03, 0.02, 0.02 and 0.02, respectively (Table 1). We observed
that only a portion of the b-LG and a-LA in the MSF were also coagulated by the addition of 0.5 M GDL. The remaining proportions of b-LG (spot 12), b-LG (spot 13) and a-LA (spot 14) in the GDL-treated MSF were 0.77, 0.73 and 0.64, respectively (Fig. 6). As previously mentioned, the addition of 0.5 M GDL to milk induces the destabilization and coagulation of casein micelles, and the isomers of aS1-CN, aS2-CN, b-CN, and k-CN, and a fraction of the bLC and a-LA, in the milk are coagulated into the pellet. The complexation between the GDL and the milk proteins yields insoluble complexes, resulting in phase separation. 3.4. Potential mechanism for the effect of GDL concentration on milk proteins Dickinson (2006) noted that the acidification process converts the liquid-like dispersion of casein particles and whey proteins into a soft, solid-like aggregated network. Our results clearly demonstrate that the total protein in the MPF increased with the addition of 0.5 M GDL, indicating that GDL reacts with caseins and a portion of whey proteins during milk coagulation. Wal (2002) reported that approximately 80% of milk protein is composed of casein, and 20% is composed of whey proteins. The results indicate that the coagulated proteins consist of the entire 80% attributed to casein and 14.3% of the total 20% of whey proteins. Based on the abovedescribed results, a reaction scheme for the effect of GDL on the coagulation of individual milk proteins is depicted in Fig. 7. The GDL-mediated coagulation reaction has been described as a twostep process. As previously mentioned, the hydrolysis of GDL to gluconic acid results in a decrease in the milk pH. The pI of aS1-CN, aS2-CN, b-CN, k-CN, b-LG and a-LA are 4.9, 4.9e5, 5.2e5.4, 5.1e5.4, 5.4e5.6, 5.2 and 4.8, respectively. The first step of the reaction scheme is a process in which GDL catalyzes the acidification of the milk. As the pH decreases, micellar phenomena occur during the entire acidification phase that precedes milk coagulation. The micellar hairy layer (k-CN) collapses, and the destabilized casein particles then aggregate together. The second step involves the GDL-mediated coagulation of the aS1-CN, aS2-CN, b-CN, and k-CN, as well as a portion of the b-LC and a-LA. However, the degree of coagulation of the whey proteins was less than that of the caseins.
Fig. 7. Reaction scheme for the effect of GDL on the coagulation of milk proteins.
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