Food Chemistry 179 (2015) 246–252
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Influence of succinylation on the conformation of yak casein micelles Min Yang a,b,c,⇑, Na Cui b,c, Yan Fang d, Ying Shi b,c, Jitao Yang a, Jiangyu Wang a a
College of Science, Gansu Agricultural University, Lanzhou, China College of Food Science and Engineering, Gansu Agricultural University, Lanzhou, China c Functional Dairy Product Engineering Lab of Gansu Province, Lanzhou, China d Instrumental Research and Analysis Center of Gansu Agricultural University, China b
a r t i c l e
i n f o
Article history: Received 2 November 2014 Received in revised form 30 January 2015 Accepted 2 February 2015 Available online 9 February 2015 Chemical compounds studied in this article: Succinic anhydride (CID: 7922) Casein (CID: 73995022) Keywords: Yak casein micelles Succinylation Conformation FTIR Fluorescence spectroscopy DLS
a b s t r a c t Succinylation modifies the physicochemical characteristics and improves the functional properties of proteins. This study assessed the effects of succinylation on the conformation of yak casein micelles with seven degree of modification. The results revealed that succinylation contributed to the dissociation of casein micelles. With the increase of succinylated degree, soluble nitrogen and minerals content increased, while casein micelle size and polydispersity index of micelles decreased. Succinylation affected the spatial conformation of yak casein micelles: turn decreased, ß-sheet and a-helix increased, and irregular structure were non-significantly affected. The intrinsic and ANS fluorescence intensity decreased and the maximum emission wavelength shifted red with increasing succinylation. Based on the results, the structure of yak casein micelles was characteristic of the sub-micelle model. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Chemical modification is commonly used to change the functional properties of food proteins (Matemu, Kayahara, Murasawa, Katayama, & Nakamura, 2011; Vidal, Marchesseau, & Cuq, 2002). Succinylation, which is one of frequently used modification method, enhances solubility, emulsification, foaming characteristics and other functional properties of protein (Gruener & Ismond, 1997; Kohara, Kanei, & Nakajima, 2001; Mirmoghtadaie, Kadivar, & Shahedi, 2009; Yang, Shi, et al., 2014). Succinylation has been shown to improve the functional properties of cow caseins (Lakkis & Villota, 1992; Strange, Holsinger, & Kleyn, 1993; Yang, Shi, et al., 2014). The functional property of a protein was strongly related to its physicochemical properties, especially its structure. Changes in some physicochemical and functional properties of proteins could be attributed to changes in nature net charge, formation or its aggregates and dissociation after succinylation (Achouri, Zhang, &
⇑ Corresponding author at: College of Science, Gansu Agricultural University, Lanzhou, China. Tel.: +86 13893272871. E-mail address: [email protected] (M. Yang). http://dx.doi.org/10.1016/j.foodchem.2015.02.003 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
Xu, 1998; Gruener & Ismond, 1997; Lawal & Adebowale, 2004; Zaghloul & Prakash, 2002). However, the effects of succinylation on protein structure stability were conflicting. Tian, Lee, Bothwell, and McGuire (1998) and van der Veen, Norde, and Stuart (2004) reported that the secondary structures of lysozyme were similar before and after succinylation. Kosters et al. (2003) reported similar circular dichroism (CD) spectra for ovalbumin modified by succinylation. In contrast, Lakkis and Villota (1992) obtained shifts in the fluorescence and CD spectra for succinylated cow casein, serum albumin (BSA), and whey protein. Achouri and Zhang (2001) reported conformational changes of soy protein hydrolysate after succinylation, which were reflected by changes in hydrophobicity and fluorescence. Knopfe et al. (1998) observed the structure changes in succinylated faba bean legumin. However, few studies have focused on the relationship between spatial structure and succinylation degree. Yak (Bos grunniens) milk is an important food source, especially in the Tibetan plateau (Li et al., 2010). Annual yak milk production has increased to 40 million tons (Li et al., 2011). Yak caseins are widely used in the production of high quality food ingredients, soaps, glues, leather polishing reagents, and clothing, among others. It has been reported that yak caseins have significantly
M. Yang et al. / Food Chemistry 179 (2015) 246–252
higher micellar calcium and lower inorganic phosphorus than cow caseins (Wang et al., 2013). Furthermore, the amino acid sequence of yak caseins is different than that of other caseins (Cui et al., 2012; Zhang et al., 2010). Additionally, yak casein micelles have different composition, size and hydration compared to cow casein micelles (Wang et al., 2013; Yang, Zhang, Wen, Zhang, & Liang, 2014). The objective of this study was to assess the effects of succinylation on the conformation of yak casein micelles. Seven succinylation levels were evaluated. The spatial structure of yak casein micelles were assessed by Fourier transform infrared spectroscopy (FTIR) and fluorescence spectroscopy. The dissociation of yak casein micelles after succinylation was detected by changes in particle size, polydispersity index, and soluble nitrogen and minerals contents. The findings of this study will provide invaluable information on the spatial structures of native and succinylated yak casein micelles.
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modification. The supernatant was used to measure soluble nitrogen and minerals contents. 2.3. Determination of the degree of modification The degree of modification was determined by the o-phthalaldehyde (OPA) method (Yang, Shi, et al., 2014). In this experiment, 3 ml of caseins solution (0.2 mg/ml) was mixed with 3 ml of OPA, which was prepared according to the method reported by Dinnella et al. (2002). After 2 min, absorbance was measured at 340 nm in a 1-cm length quartz cell (UV-2100 spectrophotometer; Beijing Beifen-Ruili Analytical Instrument Co., Ltd., Beijing, China). The number of amino groups was calculated from an L-leucine standard curve. The percentage of amino group-modified caseins was calculated using the following formula (Dinnella, Gargaro, Rossano, & Monteleone, 2002),
Modification degree ð%Þ ¼ ðN0 Nm Þ=N0 100
ð1Þ
2. Materials and methods
where N0 and Nm are the number of free amino groups in the unmodified and modified caseins, respectively.
2.1. Materials
2.4. The size and polydispersity index of casein micelles
Yak milk used in this study was collected from Tianzhu grassland, on the Qinghai-Tibetan Plateau, in northwest China. After milking, 0.02% (w/v) sodium azide was added to inhibit bacterial growth. The samples were then put in sterile plastic bottles and stored in a box filled with ice. The samples were transported to the laboratory within 6 h. Yak milk was defatted twice by centrifugation (TDD5 M, Changsha Pingfan Instrument Co. Ltd., Changsha, China) at 4000g for 10 min at 20 °C. The skim milk was centrifuged using a Beckman Optima XL-100K refrigerating ultracentrifuge (Beckman Coulter, USA) at 120,000g for 40 min at 20 °C (Françoise, Kablan, Kamenan, & Lagaude, 2009). The supernatant was removed, and the firm pellet at the bottom was the casein micelles. The pellet was freeze dried to constant weight using vacuum freeze-drying machine (GLZ-0.4, Su Yuan Zhong Tian scientific Inc., Beijing, China). Total nitrogen and casein nitrogen contents in the dried pellet were tested by the method of Li et al. (2011). Non casein nitrogen content was the difference between total nitrogen content and casein nitrogen content. It was showed that total nitrogen content in the dried pellet was 91.17 ± 0.31% (w/w) and non casein nitrogen content was 0.71 ± 0.03% (w/w).
The average size of casein micelles was determined by laser light scattering (Delsa TM Nano, Beckman Coulter Inc., CA, USA) at 25 °C and wavelength of 632.8 nm within 5 min, where the refractive index of the solvent viscosity was 1.333 and 1.002 mPa s respectively. The particles were characterized by mean diameter D and polydispersity index. The polydispersity index was obtained from the equipment directly, and D was calculated by the following equation (Wang et al., 2013):
2.2. Chemical modification of yak casein micelles Chemical modification of yak casein micelles was followed by the method of Yang, Shi, et al. (2014). Dried caseins were dissolved in distilled water at 20 mg/ml by constant mixing at 3000 rpm (40 °C). The pH of the solution was kept at 7.0 with 1 M NaOH. After caseins were completely dissolved, the pH of the solution was adjusted to 8.0–9.0 with 1 M NaOH, and the succinic anhydride was added under constant stirring for 50 min at 40 °C. Succinic anhydride was added at different succinic anhydride/ caseins ratios: 0.02:1, 0.04:1, 0.06:1, 0.08:1, 0.1:1, 0.2:1 and 0.6:1 (g/g). The pH of the solution was maintained at 8.0–9.0 with 1 M NaOH. After the reaction, the pH was adjusted to 7.0 with 1 M HCl. The solution was dialyzed against distilled water for 48 h at 4 °C in a dialysis bag (Oso-T8280, MD25, 8000–14000, Union Carbide Corporation, Danbury, USA) and centrifuged in a Beckman Optima XL-100K ultracentrifuge (rotor 70Ti; Beckman Coulter, USA) at 120,000g for 40 min at 20 °C. The solution after dialysis was employed for measurement of the spatial structure particle size, fluorescence intensity of yak caseins and the degree of
P D¼P
3 j V j Dj 2 j V j Dj
ð2Þ
where Vj is the relative volume fraction of all casein micelles with diameter Dj. Each experiment was repeated three times to acquire the average diameters of yak casein particles. 2.5. FTIR spectroscopy Casein concentrate was diluted into 10 mg/ml with deionized water. The spectroscopy of FTIR were obtained with a VERTEX 70V Bruker spectrometer (Bruker Corporation, Karlsruhe, Germany) equipped with an ATR cell (Total Attenuated Reflection mode) and a MCT (Mercury Cadmium Telluride) detector cooled with liquid N2 as described by Hussain, Gaiani, Aberkane, Ghanbaja, and Scher (2011). All data were carried out using PeakFit 4.12 software (Jandel Scientific). Raw absorbance spectra were cut between 1700 and 1600 cm1 for analyzing Amide I. Second derivative spectra were calculated on automatic baseline subtracted data using PeakFit 4.12 software. The treated spectra were deconvoluted by a nonlinear regression curve fitting program of Gaussian peaks to the original spectra until r2 > 0.999. Integrated areas of each peak were calculated and related to secondary structural features of a-helix, ß-sheet, turn and irregular structure. 2.6. Fluorescence Spectroscopy Casein concentrate was made into 2 mg/ml at pH 7.0 with deionized water. Intrinsic fluorescence experiments were performed with a RF-5301PC luminescence spectrometer (Japan Shimadzu Company) for casein solution in a 1-cm path length quartz cell at room temperature (25 °C) as reported by Liu and Guo (2008). The excitation and emission slits were fixed at 3.0
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M. Yang et al. / Food Chemistry 179 (2015) 246–252
and 1.5 nm, respectively, the excitation wavelength was set at 280 nm, and the emission spectra were collected from 290 to 450 nm. The ANS emission spectra were measured as described by Philippe, Legraet, and Gaucheron (2005). The ANS concentration was 8.0 103 M. The 200 ll ANS were mixed with 8 ml casein solution and let it stand for 3 min. The excitation and emission slits were fixed at 3.0 and 1.5 nm, respectively, the excitation wavelength was set at 390 nm, and the emission spectra were collected from 400 to 650 nm. 2.7. Determination of soluble nitrogen content Soluble nitrogen content was tested by Kjeldahl method according to the International Dairy Federation method. 2.8. Determination of minerals content Total calcium and magnesium contents in casein micelles, and soluble calcium and magnesium contents in supernatant were measured by atomic absorption spectrometry (AA-6800, Japan Shimadzu Company). Phosphorus content in casein micelles and in supernatants were determined based on the International Dairy Federation method. 2.9. Statistical analysis All data were expressed as mean ± standard deviation (SD) from at least three independent trials. The differences were assessed by one-way analysis of variance (ANOVA) and Duncan’s multiple range tests. Statistical significance was set at p < 0.05. PASW Statistics 18.0 software (SPSS Inc., Chicago, IL, US) and Origin 8.0 (OriginLab Corporation, Northampton, MA, US) were used to analyze the data. 3. Results and discussion 3.1. Modification degree The amount of modified lysine groups in caseins was dependent on the amount of succinic anhydride added. At succinic anhydride/ caseins ratios of 0.02:1, 0.04:1, 0.06:1, 0.08:1, 0.1:1, 0.2:1 and 0.6:1 (g/g), succinylation degree were determined to be 9.00 ± 0.02%, 19.38 ± 0.02%, 32.96 ± 0.03%, 44.41 ± 0.02%, 59.06 ± 0.01%, 70.21 ± 0.01% and 82.26 ± 0.01%, respectively This was comparable to the results obtained in succinylated proteins. The succinylation degree of yak and cow casein product was higher than that of yak casein micelles with the same succinic anhydride addition (Yang, Shi, et al., 2014). Casein was usually processed by acidic precipitation of milk. The structure of micelles collapsed while the minerals dissociated from micelles during caseins’ processing, resulting in caseins unfolded and a large number of lysine groups exposed to be succinylated (Chakraborty & Basak, 2007; Liu & Guo, 2008). Succinylation of proteins at similar succinic acid/protein rations depends on the protein conformation and pH value. This phenomenon has been reported in mung bean, soy, and canola proteins (Achouri & Zhang, 2001; El-Adawy, 2000; Gruener & Ismond, 1997). 3.2. Size of casein micelles Following succinylation, the average diameter of yak casein micelles decreased, (Fig. 1A). The size of native yak casein micelles after ultracentrifugation was 266.47 ± 20.47 nm, which decreased linearly to 177.83 ± 2.27 nm after succinylation with 59% degree
Fig. 1. The average size and polydispersity index of yak casein micelles with different succinylated degree.
(R2 = 0.9452), and then sharply decreased to 74.67 ± 27.00 nm with 82% succinylation degree. The size of yak casein micelles obtained by ultracentrifugation was larger than that in yak milk, which was 211.3 nm (Yang, Zhang, et al., 2014). It has been reported that the size of casein micelles became more slim with increase of pH whereas it also increased after heat treatment (Erdem, 2006; Yang, Zhang, et al., 2014). Although the size of casein micelles reduced by ultracentrifugation (Sandra & Dalgleish, 2005), it became loose under heat treatment at 40 °C with pH 7.0, higher than native pH of milk (6.7), which induced the increase of micellar size. On the other hand, the concentrate of micelles in the samples was lower than that in milk, contributing to larger yak casein micelles following ultracentrifugation. Similarly, Vidal, Marchesseau, and Cuq (2002) reported a reduction in casein micellar size after succinylation. According to the sub-micelle model (Walstra, 1999), succinylation occurred first on j-caseins located on micelle surface, increasing the negative charges and the electrostatic repulsions between j-caseins and other casein fractions in sub-micelle. As a result, j-caseins dissociated from the casein micelles. Subsequently, succinylation occurred on casein molecules located on the surface of sub-micelles at the outer edge of particles, leading to increased repulsion and steric effects, which caused the dissociation of phosphate groups, disaggregation of outer sub-micelles, and dissociation of succinylated casein molecules from sub-micelles. With the degree of modification increasing, sub-micelles were dissociated outside-in, meanwhile the phosphate dissociated. At succinylation >59%, a large number of sub-micelles disaggregated and micellar size decreased sharply. The polydispersity indexes (P) were shown in Fig. 1B. The polydispersity indexes are interpreted as follows: 0 6 P 6 0.02, which is characteristic of monodisperse or nearly monodisperse systems; 0.02 < P 6 0.08, which is characteristic of narrow particle size distributions; and P > 0.08, which indicates broader size distributions (Chappellaz, Alexander, & Corredig, 2010). According to Fig. 1B, the polydispersity index of yak casein micelles decreased with increasing succinylation degree. Yak casein micelles had a broader particle size distributions (P > 0.08) after ultracentrifugation, and a narrow particle size distributions after succinylation >19% (P < 0.08). At 82% succinylation, P was 0.02, which revealed that casein micelles were in a monodisperse system. Casein micelles in yak milk had narrow particle size distributions (P < 0.08) (Yang, Shi, et al., 2014). By ultracentrifugation, casein micelles aggregated, solubility became poor, and P was
M. Yang et al. / Food Chemistry 179 (2015) 246–252
higher than it in milk. The reduction in P was attributed to an increase in casein solubility and to a reduction in casein micellar size after succinylation (Yang, Shi, et al., 2014).
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differences in the amino acid sequence from different bovine breeds (Bai et al., 2010). 3.4. Fluorescence Spectroscopy of yak caseins
3.3. FTIR spectra of yak casein micelles FTIR spectra of yak casein micelles with and without succinylation in water at pH 7.0 were recorded as described in the experimental section. Second derivative spectra were calculated from Amide I regions. The spatial structures content of yak casein micelles were shown in Fig. 2. Peak assignment of Amide I region was performed using the results of former studies for caseins (Hussain et al., 2011). During the iteration process the peaks positions were allowed to shift which was less than 2 cm1. For Amide I regions, the following structures were assigned: 1656–1700 cm1, turn; 1650– 1655 cm1, a-helix; 1640–1649 cm1, irregular; and 1600– 1639 cm1, ß-sheet structure. The secondary structure content of yak casein micelles was calculated by summing the areas of the FTIR bands. The results revealed a considerable amount of ß-sheet and turn. The content of turn decreased following 9–59% succinylation; however the results were not significant (p > 0.05). The content of ß-sheet increased after 9–44% and 33–82% succinylation (p > 0.05). The content of a-helix increased after 9–82% and 44–82% succinylation (p > 0.05). The content of irregular increased with succinylation level (p > 0.05). Bovine caseins have never been crystallized; therefore, there was little information on their structures (Hussain et al., 2011). Nevertheless, tentative assignments of secondary structural components in bovine caseins have been performed (Curley, Kumosinski, Unruh, & Farrell, 1998). First secondary structural elements of yak caseins were identified by FTIR. The results revealed that there were 36.42 ± 1.84% ß–sheet, 35.33 ± 2.04% turn, 18.37 ± 2.53% irregular and 9.88 ± 1.95% a-helix. Hussain et al. (2011) reported that 5% cow casein micelles in water (w/w) contain 12% a-helix, 45% ß-sheet, 26% turn, and 16% irregular structure for FTIR amide I. The results were probably attributed to
Intrinsic fluorescence emission spectra of yak casein micelles with different modified degree of excited at 280 nm were shown in Fig. 3A. Fig. 3B showed the effects of succinylation on fluorescence maximum intensity and the maximum emission wavelength (kmax) of tryptophan residues. With increasing succinylation, there was a progressive red shift of kmax with 12 nm while the emission intensity decreased. The kmax value is very useful for estimating the hydrophobicity of the micro environment around tryptophan residues. At 335– 350 nm kmax, tryptophan residues are buried in a hydrophobic domain of the protein, that is they are located in the less polar region; at 350–353 nm kmax, tryptophan residues are exposed to water (Liu & Guo, 2008). In intact yak casein micelles, kmax was 336 nm, suggesting that the tryptophan residues were located in a more hydrophobic domain. During succinylation, the negatively charged succinyl groups and the deprotonation of the carboxylic groups changed the conformation of casein micelles, by favoring electrostatic repulsions and hydrogen bonds, salt bridges destruction, and loose casein micelles structures, which exposed more tryptophan residues to the polar environment (Vidal, Marchesseau, Lagaude, & Cuq, 1998). At succinylation
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Influence of succinylation on the conformation of yak casein micelles Min Yang a,b,c,⇑, Na Cui b,c, Yan Fang d, Ying Shi b,c, Jitao Yang a, Jiangyu Wang a a
College of Science, Gansu Agricultural University, Lanzhou, China College of Food Science and Engineering, Gansu Agricultural University, Lanzhou, China c Functional Dairy Product Engineering Lab of Gansu Province, Lanzhou, China d Instrumental Research and Analysis Center of Gansu Agricultural University, China b
a r t i c l e
i n f o
Article history: Received 2 November 2014 Received in revised form 30 January 2015 Accepted 2 February 2015 Available online 9 February 2015 Chemical compounds studied in this article: Succinic anhydride (CID: 7922) Casein (CID: 73995022) Keywords: Yak casein micelles Succinylation Conformation FTIR Fluorescence spectroscopy DLS
a b s t r a c t Succinylation modifies the physicochemical characteristics and improves the functional properties of proteins. This study assessed the effects of succinylation on the conformation of yak casein micelles with seven degree of modification. The results revealed that succinylation contributed to the dissociation of casein micelles. With the increase of succinylated degree, soluble nitrogen and minerals content increased, while casein micelle size and polydispersity index of micelles decreased. Succinylation affected the spatial conformation of yak casein micelles: turn decreased, ß-sheet and a-helix increased, and irregular structure were non-significantly affected. The intrinsic and ANS fluorescence intensity decreased and the maximum emission wavelength shifted red with increasing succinylation. Based on the results, the structure of yak casein micelles was characteristic of the sub-micelle model. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Chemical modification is commonly used to change the functional properties of food proteins (Matemu, Kayahara, Murasawa, Katayama, & Nakamura, 2011; Vidal, Marchesseau, & Cuq, 2002). Succinylation, which is one of frequently used modification method, enhances solubility, emulsification, foaming characteristics and other functional properties of protein (Gruener & Ismond, 1997; Kohara, Kanei, & Nakajima, 2001; Mirmoghtadaie, Kadivar, & Shahedi, 2009; Yang, Shi, et al., 2014). Succinylation has been shown to improve the functional properties of cow caseins (Lakkis & Villota, 1992; Strange, Holsinger, & Kleyn, 1993; Yang, Shi, et al., 2014). The functional property of a protein was strongly related to its physicochemical properties, especially its structure. Changes in some physicochemical and functional properties of proteins could be attributed to changes in nature net charge, formation or its aggregates and dissociation after succinylation (Achouri, Zhang, &
⇑ Corresponding author at: College of Science, Gansu Agricultural University, Lanzhou, China. Tel.: +86 13893272871. E-mail address: [email protected] (M. Yang). http://dx.doi.org/10.1016/j.foodchem.2015.02.003 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
Xu, 1998; Gruener & Ismond, 1997; Lawal & Adebowale, 2004; Zaghloul & Prakash, 2002). However, the effects of succinylation on protein structure stability were conflicting. Tian, Lee, Bothwell, and McGuire (1998) and van der Veen, Norde, and Stuart (2004) reported that the secondary structures of lysozyme were similar before and after succinylation. Kosters et al. (2003) reported similar circular dichroism (CD) spectra for ovalbumin modified by succinylation. In contrast, Lakkis and Villota (1992) obtained shifts in the fluorescence and CD spectra for succinylated cow casein, serum albumin (BSA), and whey protein. Achouri and Zhang (2001) reported conformational changes of soy protein hydrolysate after succinylation, which were reflected by changes in hydrophobicity and fluorescence. Knopfe et al. (1998) observed the structure changes in succinylated faba bean legumin. However, few studies have focused on the relationship between spatial structure and succinylation degree. Yak (Bos grunniens) milk is an important food source, especially in the Tibetan plateau (Li et al., 2010). Annual yak milk production has increased to 40 million tons (Li et al., 2011). Yak caseins are widely used in the production of high quality food ingredients, soaps, glues, leather polishing reagents, and clothing, among others. It has been reported that yak caseins have significantly
M. Yang et al. / Food Chemistry 179 (2015) 246–252
higher micellar calcium and lower inorganic phosphorus than cow caseins (Wang et al., 2013). Furthermore, the amino acid sequence of yak caseins is different than that of other caseins (Cui et al., 2012; Zhang et al., 2010). Additionally, yak casein micelles have different composition, size and hydration compared to cow casein micelles (Wang et al., 2013; Yang, Zhang, Wen, Zhang, & Liang, 2014). The objective of this study was to assess the effects of succinylation on the conformation of yak casein micelles. Seven succinylation levels were evaluated. The spatial structure of yak casein micelles were assessed by Fourier transform infrared spectroscopy (FTIR) and fluorescence spectroscopy. The dissociation of yak casein micelles after succinylation was detected by changes in particle size, polydispersity index, and soluble nitrogen and minerals contents. The findings of this study will provide invaluable information on the spatial structures of native and succinylated yak casein micelles.
247
modification. The supernatant was used to measure soluble nitrogen and minerals contents. 2.3. Determination of the degree of modification The degree of modification was determined by the o-phthalaldehyde (OPA) method (Yang, Shi, et al., 2014). In this experiment, 3 ml of caseins solution (0.2 mg/ml) was mixed with 3 ml of OPA, which was prepared according to the method reported by Dinnella et al. (2002). After 2 min, absorbance was measured at 340 nm in a 1-cm length quartz cell (UV-2100 spectrophotometer; Beijing Beifen-Ruili Analytical Instrument Co., Ltd., Beijing, China). The number of amino groups was calculated from an L-leucine standard curve. The percentage of amino group-modified caseins was calculated using the following formula (Dinnella, Gargaro, Rossano, & Monteleone, 2002),
Modification degree ð%Þ ¼ ðN0 Nm Þ=N0 100
ð1Þ
2. Materials and methods
where N0 and Nm are the number of free amino groups in the unmodified and modified caseins, respectively.
2.1. Materials
2.4. The size and polydispersity index of casein micelles
Yak milk used in this study was collected from Tianzhu grassland, on the Qinghai-Tibetan Plateau, in northwest China. After milking, 0.02% (w/v) sodium azide was added to inhibit bacterial growth. The samples were then put in sterile plastic bottles and stored in a box filled with ice. The samples were transported to the laboratory within 6 h. Yak milk was defatted twice by centrifugation (TDD5 M, Changsha Pingfan Instrument Co. Ltd., Changsha, China) at 4000g for 10 min at 20 °C. The skim milk was centrifuged using a Beckman Optima XL-100K refrigerating ultracentrifuge (Beckman Coulter, USA) at 120,000g for 40 min at 20 °C (Françoise, Kablan, Kamenan, & Lagaude, 2009). The supernatant was removed, and the firm pellet at the bottom was the casein micelles. The pellet was freeze dried to constant weight using vacuum freeze-drying machine (GLZ-0.4, Su Yuan Zhong Tian scientific Inc., Beijing, China). Total nitrogen and casein nitrogen contents in the dried pellet were tested by the method of Li et al. (2011). Non casein nitrogen content was the difference between total nitrogen content and casein nitrogen content. It was showed that total nitrogen content in the dried pellet was 91.17 ± 0.31% (w/w) and non casein nitrogen content was 0.71 ± 0.03% (w/w).
The average size of casein micelles was determined by laser light scattering (Delsa TM Nano, Beckman Coulter Inc., CA, USA) at 25 °C and wavelength of 632.8 nm within 5 min, where the refractive index of the solvent viscosity was 1.333 and 1.002 mPa s respectively. The particles were characterized by mean diameter D and polydispersity index. The polydispersity index was obtained from the equipment directly, and D was calculated by the following equation (Wang et al., 2013):
2.2. Chemical modification of yak casein micelles Chemical modification of yak casein micelles was followed by the method of Yang, Shi, et al. (2014). Dried caseins were dissolved in distilled water at 20 mg/ml by constant mixing at 3000 rpm (40 °C). The pH of the solution was kept at 7.0 with 1 M NaOH. After caseins were completely dissolved, the pH of the solution was adjusted to 8.0–9.0 with 1 M NaOH, and the succinic anhydride was added under constant stirring for 50 min at 40 °C. Succinic anhydride was added at different succinic anhydride/ caseins ratios: 0.02:1, 0.04:1, 0.06:1, 0.08:1, 0.1:1, 0.2:1 and 0.6:1 (g/g). The pH of the solution was maintained at 8.0–9.0 with 1 M NaOH. After the reaction, the pH was adjusted to 7.0 with 1 M HCl. The solution was dialyzed against distilled water for 48 h at 4 °C in a dialysis bag (Oso-T8280, MD25, 8000–14000, Union Carbide Corporation, Danbury, USA) and centrifuged in a Beckman Optima XL-100K ultracentrifuge (rotor 70Ti; Beckman Coulter, USA) at 120,000g for 40 min at 20 °C. The solution after dialysis was employed for measurement of the spatial structure particle size, fluorescence intensity of yak caseins and the degree of
P D¼P
3 j V j Dj 2 j V j Dj
ð2Þ
where Vj is the relative volume fraction of all casein micelles with diameter Dj. Each experiment was repeated three times to acquire the average diameters of yak casein particles. 2.5. FTIR spectroscopy Casein concentrate was diluted into 10 mg/ml with deionized water. The spectroscopy of FTIR were obtained with a VERTEX 70V Bruker spectrometer (Bruker Corporation, Karlsruhe, Germany) equipped with an ATR cell (Total Attenuated Reflection mode) and a MCT (Mercury Cadmium Telluride) detector cooled with liquid N2 as described by Hussain, Gaiani, Aberkane, Ghanbaja, and Scher (2011). All data were carried out using PeakFit 4.12 software (Jandel Scientific). Raw absorbance spectra were cut between 1700 and 1600 cm1 for analyzing Amide I. Second derivative spectra were calculated on automatic baseline subtracted data using PeakFit 4.12 software. The treated spectra were deconvoluted by a nonlinear regression curve fitting program of Gaussian peaks to the original spectra until r2 > 0.999. Integrated areas of each peak were calculated and related to secondary structural features of a-helix, ß-sheet, turn and irregular structure. 2.6. Fluorescence Spectroscopy Casein concentrate was made into 2 mg/ml at pH 7.0 with deionized water. Intrinsic fluorescence experiments were performed with a RF-5301PC luminescence spectrometer (Japan Shimadzu Company) for casein solution in a 1-cm path length quartz cell at room temperature (25 °C) as reported by Liu and Guo (2008). The excitation and emission slits were fixed at 3.0
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and 1.5 nm, respectively, the excitation wavelength was set at 280 nm, and the emission spectra were collected from 290 to 450 nm. The ANS emission spectra were measured as described by Philippe, Legraet, and Gaucheron (2005). The ANS concentration was 8.0 103 M. The 200 ll ANS were mixed with 8 ml casein solution and let it stand for 3 min. The excitation and emission slits were fixed at 3.0 and 1.5 nm, respectively, the excitation wavelength was set at 390 nm, and the emission spectra were collected from 400 to 650 nm. 2.7. Determination of soluble nitrogen content Soluble nitrogen content was tested by Kjeldahl method according to the International Dairy Federation method. 2.8. Determination of minerals content Total calcium and magnesium contents in casein micelles, and soluble calcium and magnesium contents in supernatant were measured by atomic absorption spectrometry (AA-6800, Japan Shimadzu Company). Phosphorus content in casein micelles and in supernatants were determined based on the International Dairy Federation method. 2.9. Statistical analysis All data were expressed as mean ± standard deviation (SD) from at least three independent trials. The differences were assessed by one-way analysis of variance (ANOVA) and Duncan’s multiple range tests. Statistical significance was set at p < 0.05. PASW Statistics 18.0 software (SPSS Inc., Chicago, IL, US) and Origin 8.0 (OriginLab Corporation, Northampton, MA, US) were used to analyze the data. 3. Results and discussion 3.1. Modification degree The amount of modified lysine groups in caseins was dependent on the amount of succinic anhydride added. At succinic anhydride/ caseins ratios of 0.02:1, 0.04:1, 0.06:1, 0.08:1, 0.1:1, 0.2:1 and 0.6:1 (g/g), succinylation degree were determined to be 9.00 ± 0.02%, 19.38 ± 0.02%, 32.96 ± 0.03%, 44.41 ± 0.02%, 59.06 ± 0.01%, 70.21 ± 0.01% and 82.26 ± 0.01%, respectively This was comparable to the results obtained in succinylated proteins. The succinylation degree of yak and cow casein product was higher than that of yak casein micelles with the same succinic anhydride addition (Yang, Shi, et al., 2014). Casein was usually processed by acidic precipitation of milk. The structure of micelles collapsed while the minerals dissociated from micelles during caseins’ processing, resulting in caseins unfolded and a large number of lysine groups exposed to be succinylated (Chakraborty & Basak, 2007; Liu & Guo, 2008). Succinylation of proteins at similar succinic acid/protein rations depends on the protein conformation and pH value. This phenomenon has been reported in mung bean, soy, and canola proteins (Achouri & Zhang, 2001; El-Adawy, 2000; Gruener & Ismond, 1997). 3.2. Size of casein micelles Following succinylation, the average diameter of yak casein micelles decreased, (Fig. 1A). The size of native yak casein micelles after ultracentrifugation was 266.47 ± 20.47 nm, which decreased linearly to 177.83 ± 2.27 nm after succinylation with 59% degree
Fig. 1. The average size and polydispersity index of yak casein micelles with different succinylated degree.
(R2 = 0.9452), and then sharply decreased to 74.67 ± 27.00 nm with 82% succinylation degree. The size of yak casein micelles obtained by ultracentrifugation was larger than that in yak milk, which was 211.3 nm (Yang, Zhang, et al., 2014). It has been reported that the size of casein micelles became more slim with increase of pH whereas it also increased after heat treatment (Erdem, 2006; Yang, Zhang, et al., 2014). Although the size of casein micelles reduced by ultracentrifugation (Sandra & Dalgleish, 2005), it became loose under heat treatment at 40 °C with pH 7.0, higher than native pH of milk (6.7), which induced the increase of micellar size. On the other hand, the concentrate of micelles in the samples was lower than that in milk, contributing to larger yak casein micelles following ultracentrifugation. Similarly, Vidal, Marchesseau, and Cuq (2002) reported a reduction in casein micellar size after succinylation. According to the sub-micelle model (Walstra, 1999), succinylation occurred first on j-caseins located on micelle surface, increasing the negative charges and the electrostatic repulsions between j-caseins and other casein fractions in sub-micelle. As a result, j-caseins dissociated from the casein micelles. Subsequently, succinylation occurred on casein molecules located on the surface of sub-micelles at the outer edge of particles, leading to increased repulsion and steric effects, which caused the dissociation of phosphate groups, disaggregation of outer sub-micelles, and dissociation of succinylated casein molecules from sub-micelles. With the degree of modification increasing, sub-micelles were dissociated outside-in, meanwhile the phosphate dissociated. At succinylation >59%, a large number of sub-micelles disaggregated and micellar size decreased sharply. The polydispersity indexes (P) were shown in Fig. 1B. The polydispersity indexes are interpreted as follows: 0 6 P 6 0.02, which is characteristic of monodisperse or nearly monodisperse systems; 0.02 < P 6 0.08, which is characteristic of narrow particle size distributions; and P > 0.08, which indicates broader size distributions (Chappellaz, Alexander, & Corredig, 2010). According to Fig. 1B, the polydispersity index of yak casein micelles decreased with increasing succinylation degree. Yak casein micelles had a broader particle size distributions (P > 0.08) after ultracentrifugation, and a narrow particle size distributions after succinylation >19% (P < 0.08). At 82% succinylation, P was 0.02, which revealed that casein micelles were in a monodisperse system. Casein micelles in yak milk had narrow particle size distributions (P < 0.08) (Yang, Shi, et al., 2014). By ultracentrifugation, casein micelles aggregated, solubility became poor, and P was
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higher than it in milk. The reduction in P was attributed to an increase in casein solubility and to a reduction in casein micellar size after succinylation (Yang, Shi, et al., 2014).
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differences in the amino acid sequence from different bovine breeds (Bai et al., 2010). 3.4. Fluorescence Spectroscopy of yak caseins
3.3. FTIR spectra of yak casein micelles FTIR spectra of yak casein micelles with and without succinylation in water at pH 7.0 were recorded as described in the experimental section. Second derivative spectra were calculated from Amide I regions. The spatial structures content of yak casein micelles were shown in Fig. 2. Peak assignment of Amide I region was performed using the results of former studies for caseins (Hussain et al., 2011). During the iteration process the peaks positions were allowed to shift which was less than 2 cm1. For Amide I regions, the following structures were assigned: 1656–1700 cm1, turn; 1650– 1655 cm1, a-helix; 1640–1649 cm1, irregular; and 1600– 1639 cm1, ß-sheet structure. The secondary structure content of yak casein micelles was calculated by summing the areas of the FTIR bands. The results revealed a considerable amount of ß-sheet and turn. The content of turn decreased following 9–59% succinylation; however the results were not significant (p > 0.05). The content of ß-sheet increased after 9–44% and 33–82% succinylation (p > 0.05). The content of a-helix increased after 9–82% and 44–82% succinylation (p > 0.05). The content of irregular increased with succinylation level (p > 0.05). Bovine caseins have never been crystallized; therefore, there was little information on their structures (Hussain et al., 2011). Nevertheless, tentative assignments of secondary structural components in bovine caseins have been performed (Curley, Kumosinski, Unruh, & Farrell, 1998). First secondary structural elements of yak caseins were identified by FTIR. The results revealed that there were 36.42 ± 1.84% ß–sheet, 35.33 ± 2.04% turn, 18.37 ± 2.53% irregular and 9.88 ± 1.95% a-helix. Hussain et al. (2011) reported that 5% cow casein micelles in water (w/w) contain 12% a-helix, 45% ß-sheet, 26% turn, and 16% irregular structure for FTIR amide I. The results were probably attributed to
Intrinsic fluorescence emission spectra of yak casein micelles with different modified degree of excited at 280 nm were shown in Fig. 3A. Fig. 3B showed the effects of succinylation on fluorescence maximum intensity and the maximum emission wavelength (kmax) of tryptophan residues. With increasing succinylation, there was a progressive red shift of kmax with 12 nm while the emission intensity decreased. The kmax value is very useful for estimating the hydrophobicity of the micro environment around tryptophan residues. At 335– 350 nm kmax, tryptophan residues are buried in a hydrophobic domain of the protein, that is they are located in the less polar region; at 350–353 nm kmax, tryptophan residues are exposed to water (Liu & Guo, 2008). In intact yak casein micelles, kmax was 336 nm, suggesting that the tryptophan residues were located in a more hydrophobic domain. During succinylation, the negatively charged succinyl groups and the deprotonation of the carboxylic groups changed the conformation of casein micelles, by favoring electrostatic repulsions and hydrogen bonds, salt bridges destruction, and loose casein micelles structures, which exposed more tryptophan residues to the polar environment (Vidal, Marchesseau, Lagaude, & Cuq, 1998). At succinylation
